Multi-drug ligand conjugates

ABSTRACT

Described herein are compounds, pharmaceutical compositions and methods for treating pathogenic cell populations in a patient. The compounds described herein include conjugates of a plurality of cytotoxic drugs and vitamin receptor binding ligands. The plurality of drugs may be the same or different. Similarly, the vitamin receptor binding ligands may be the same or different. The conjugates also include a linker that is formed from one or more spacer linkers, heteroatom linkers, and releasable linkers.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional patent application Ser. No. 60/709,950, filed Aug. 19, 2005,and U.S. provisional patent application Ser. No. 60/787,558, filed Mar.30, 2006, the entirety of the disclosures of which are incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to compositions and methods for use intargeted drug delivery. In particular, the invention relates to ligandconjugates including two or more drugs, and analogs and derivativesthereof, such as conjugates of vitamin receptor binding compounds andtwo or more drugs.

BACKGROUND

The mammalian immune system provides a means for the recognition andelimination of tumor cells, other pathogenic cells, and invading foreignpathogens. While the immune system normally provides a strong line ofdefense, there are many instances where cancer cells, other pathogeniccells, or infectious agents evade a host immune response and proliferateor persist with concomitant host pathogenicity. Chemotherapeutic agentsand radiation therapies have been developed to eliminate, for example,replicating neoplasms. However, many of the currently availablechemotherapeutic agents and radiation therapy regimens have adverse sideeffects because they work not only to destroy pathogenic cells, but theyalso affect normal host cells, such as cells of the hematopoieticsystem. The adverse side effects of these anticancer drugs highlight theneed for the development of new therapies selective for pathogenic cellpopulations and with reduced host toxicity.

Researchers have developed therapeutic protocols for destroyingpathogenic cells by targeting cytotoxic compounds to such cells. Many ofthese protocols utilize toxins conjugated to antibodies that bind toantigens unique to or overexpressed by the pathogenic cells in anattempt to minimize delivery of the toxin to normal cells. Using thisapproach, certain immunotoxins have been developed consisting ofantibodies directed to specific antigens on pathogenic cells, theantibodies being linked to toxins such as ricin, Pseudomonas exotoxin,Diptheria toxin, and tumor necrosis factor. These immunotoxins targetpathogenic cells, such as tumor cells, bearing the specific antigensrecognized by the antibody (Olsnes, S., Immunol. Today, 10, pp. 291-295,1989; Melby, E L., Cancer Res., 53(8), pp. 1755-1760, 1993; Better, M.D., PCT International Publication no. WO 91/07418, published May 30,1991).

Another approach for targeting populations of pathogenic cells, such ascancer cells or foreign pathogens, in a host is to enhance the hostimmune response against the pathogenic cells to avoid the need foradministration of compounds that may also exhibit independent hosttoxicity. One reported strategy for immunotherapy is to bind antibodies,for example, genetically engineered multimeric antibodies, to thesurface of tumor cells to display the constant region of the antibodieson the cell surface and thereby induce tumor cell killing by variousimmune-system mediated processes (De Vita, V. T., Biologic Therapy ofCancer, 2d ed. Philadelphia, Lippincott, 1995; Soulillou, L P., U.S.Pat. No. 5,672,486). However, these approaches have been complicated bythe difficulties in defining tumor-specific antigens.

SUMMARY OF THE INVENTION

Ligand conjugates of drugs, and analogs and derivatives thereof, aredescribed herein. The conjugates include cell receptor binding ligandsthat are covalently attached to two or more drugs that may be targetedto cells. The conjugates described herein may also include a polyvalentlinker for attaching the ligands to the drugs.

In one embodiment, a receptor binding drug delivery conjugate isdescribed. The drug delivery conjugate comprises a ligand of a cellsurface receptor, two or more drugs, or analogs or derivatives thereof,and optionally a polyvalent linker, which may be generally representedby the formula

(B)-(L)-(D)_(n)

wherein (B) represents a receptor binding moiety; (D) represents a drug,or analog or derivative thereof, to be targeted to a cell by thereceptor binding moiety; (L) represents a polyvalent linker, and n is aninteger greater than 1. The polyvalent linker (L) can comprise multiplelinkers covalently attached to each other. For example, the polyvalentlinker (L) can comprise one or more spacer linkers (l_(s)), and/orreleasable linkers (l_(r)), each connected to the other, and to theligand and the drug, by one or more heteroatom linkers (l_(H)). Thesevarious linkers may be selected and placed in any order to construct thepolyvalent linker (L). Illustratively, the polyvalent linker (L) may beconstructed from one or more of the following bivalent linkers:

-(L)-

-(l_(r))_(c)-

-(l_(s))_(a-)

-(l_(s))_(a)-(l_(r))_(c)-

-(l_(r))_(c)-(l_(s))_(a)-

-(l_(H))_(b)-(l_(r))_(c)-

-(l_(r))_(c)-(l_(H))_(b)-

-(l_(H))_(d)-(l_(r))_(c)-(l_(H))_(e)-

-(l_(s))_(a)-(l_(H))_(b)-(l_(r))_(c)-

-(l_(r))_(c)-(l_(H))_(b)-(l_(s))_(a)-

-(l_(H))_(d)-(l_(s))_(a)-(l_(r))_(c)-(l_(H))_(e)-

-(l_(H))_(d)-(l_(r))_(c)-(l_(s))_(a)-(l_(H))_(e)-

-(l_(H))_(d)-(l_(s))_(a)-(l_(H))_(b)-(l_(r))_(c)-(l_(H))_(e)-

-(l_(H))_(d)-(l_(r))_(c)-(l_(H))_(b)-(l_(s))_(a)-(l_(H))_(e)-

-(l_(s))_(a)-(l_(r))_(c)-(l_(H))_(b)-

-[(l_(s))_(a)-(l_(H))_(b)]_(d)-(l_(r))_(c)-(l_(H))_(e)-

wherein a, b, c, d, and e are integers, such as integers in the rangefrom 0 to about 4, and (l_(s)), (l_(H)), and (l_(r)) are the spacerlinkers, releasable linkers, heteroatom linkers, respectively.Additional illustrative examples of bivalent linkers that may be used toconstruct the polyvalent linkers described herein are described in U.S.patent application Ser. No. 10/765,336 (also found as U.S. patentapplication publication no. US 2005/0002942 A1) and PCT internationalpublication no. WO 2006/012527, the entirety of the disclosures of whichare incorporated herein by reference.

It is to be understood that the polyvalent linkers may connect thereceptor binding moiety to the two or more drugs in a variety ofstructural configurations, including but not limited to the followingillustrative general formulae:

where B is the receptor binding ligand, each of (L¹), (L²), and (L³) isa polyvalent linker constructed from one or more spacer, releasable,and/or heteroatom linkers, and each of (D¹), D², and D³ is a drug, or ananalog or derivative thereof. Other variations, including additionaldrugs, or analogs or derivatives thereof, additional linkers, andadditional configurations of the arrangement of each of (B), (L), and(D), are also contemplated herein.

In one variation, more than one receptor binding ligand is included inthe drug delivery conjugates described herein, including but not limitedto the following illustrative general formulae:

where each B is a receptor binding ligand, each of (L¹), (L²), and (L³)is a polyvalent linker constructed from one or more spacer, releasable,and/or heteroatom linkers, and each of (D¹), D², and D³ is a drug, or ananalog or derivative thereof. Other variations, including additionaldrugs, or analogs or derivatives thereof, additional linkers, andadditional configurations of the arrangement of each of (B), (L), and(D), are also contemplated herein. In one variation, the receptorbinding ligands are for the same receptor, and in another variation, thereceptor binding ligands are for different receptors.

In one illustrative embodiment of the drug delivery conjugates describedherein, the polyvalent linker includes at least one releasable linker(I_(r)). In another illustrative embodiment of the drug deliveryconjugates described herein, the polyvalent linker includes at least tworeleasable linkers (l₂)₂. In another illustrative aspect, the polyvalentlinker (L) includes at least one releasable linkers (l_(r)) that is nota disulfide releasable linker. In another illustrative aspect, thepolyvalent linker (L) has at least two releasable linkers (l_(r))₂ whereone releasable linker is not a disulfide releasable linker. It isappreciated that when more than one releasable linker is included in thepolyvalent linker, those releasable linkers may be adjacent. It isfurther appreciated that when two releasable linkers are adjacent in thepolyvalent linker, the two releasable linkers may cooperate to causerelease of the drug.

In another embodiment, the polyvalent linker includes at least onespacer linker that is a peptide formed from amino acids. In one aspect,the peptide includes naturally occurring amino acids, and stereoisomersthereof. In another aspect, the peptide is formed only from naturallyoccurring amino acids, and stereoisomers thereof.

The ligands described herein generally include ligands of cell surfacereceptors. Illustrative ligands useful in the conjugates describedherein include, but are not limited to, vitamins, and other moietiesthat bind to a vitamin receptor, transporter, or other surface-presentedprotein that specifically binds vitamins, or analogs or derivativesthereof, peptide ligands identified from library screens, tumorcell-specific peptides, tumor cell-specific aptamers, tumorcell-specific carbohydrates, tumor cell-specific monoclonal orpolyclonal antibodies, Fab or scFv (i.e., a single chain variableregion) fragments of antibodies such as, for example, an Fab fragment ofan antibody directed to EphA2 or other proteins specifically expressedor uniquely accessible on metastatic cancer cells, small organicmolecules derived from combinatorial libraries, growth factors, such asEGF, FGF, insulin, and insulin-like growth factors, and homologouspolypeptides, somatostatin and its analogs, transferrin, lipoproteincomplexes, bile salts, selectins, steroid hormones, Arg-Gly-Aspcontaining peptides, retinoids, various Galectins, δ-opioid receptorligands, cholecystokinin A receptor ligands, ligands specific forangiotensin AT1 or AT2 receptors, peroxisome proliferator-activatedreceptor λ ligands, β-lactam antibiotics such as penicillin, smallorganic molecules including antimicrobial drugs, and other moleculesthat bind specifically to a receptor preferentially expressed on thesurface of tumor cells or on an infectious organism, antimicrobial andother drugs designed to fit into the binding pocket of a particularreceptor based on the crystal structure of the receptor or other cellsurface protein, ligands of tumor antigens or other moleculespreferentially expressed on the surface of tumor cells, or fragments ofany of these molecules. Tumor-specific antigens that could function as abinding site for ligand-drug conjugates include extracellular epitopesof members of the Ephrin family of proteins, such as EphA2. EphA2expression is restricted to cell-cell junctions in normal cells, butEphA2 is distributed over the entire cell surface in metastatic tumorcells. Thus, EphA2 on metastatic cells would be accessible for bindingto, for example, an Fab fragment of an antibody conjugated to a drug, oranalog or derivative thereof, whereas the protein would not beaccessible for binding to the Fab fragment on normal cells, resulting ina ligand-drug conjugate specific for metastatic cancer cells.

The drugs, and various analogs and derivatives thereof, described hereinare generally drugs for eliminating, killing, interfering with, and/ordecreasing the growth of a population of pathogenic cells, includinginfectious agents, cancers, tumors, and the like. Further, the drugs,and the various analogs and derivatives thereof, useful in theconjugates described herein may have a wide variety of mechanisms ofaction, including but not limited to alkylating agents, microtubuleinhibitors, including those that stabilize and/or destabilizemicrotubule formation, including beta-tubulin agents, cyclin dependentkinase (CDK) inhibitors such as CDKN1a. CDKN1b, and the like,topoisomerase inhibitors, protein synthesis inhibitors, protein kinaseinhibitors, including Ras, Raf, PKC, PI3K, and like inhibitors,transcription inhibitor, antifolates, heat shock protein blockers, andthe like.

In another embodiment, a pharmaceutical composition is described. Thepharmaceutical composition comprises a drug delivery conjugate describedherein in combination with a pharmaceutically acceptable carrier,excipient, and/or diluent therefor.

In another embodiment, a method for eliminating a population ofpathogenic cells in a host animal harboring the population of pathogeniccells is described. In one illustrative aspect, the members of thepathogenic cell population have an accessible binding site for areceptor binding moiety, or the analog or derivative thereof, and thatbinding site is uniquely expressed, overexpressed, or preferentiallyexpressed by the pathogenic cells. The method includes the step ofadministering to the host a drug delivery conjugate described herein, ora pharmaceutical composition thereof, as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the relative binding affinity of Example 9 (▪, 0.24)versus folic acid (, 1.0) at folic acid receptors.

FIG. 1B shows the activity of Example 9 on ³H-thymidine incorporation inKB cells with (∘) and without () excess folic acid; IC₅₀ of Example 9is about 58 nM.

FIG. 2 shows the relative binding affinity of for Example 11 (▪, 0.21)versus folic acid (, 1.0) at folic acid receptors.

FIG. 3 shows the activity of Example 11 (multi-drug conjugate) on³H-thymidine incorporation with (∘) and without () excess folic acid;IC₅₀ of Example 11=5 nM.

FIG. 4 shows the in vitro cytotoxic activity of Example 11 (a) on threedifferent tumor cell lines (KB, 4T-1cl2, and ID8-cl15) compared toExample 11+ excess folic acid (b).

FIG. 5A shows the activity of Example 11 at 1 μmol/kg TIW (6 doses) (),and 2 μmol/kg TIW (6 doses) (▾) on FR-positive M109 tumors in Balb/cmice versus untreated controls (▪).

FIG. 5B shows the absence of an effect by Example 11 at 1 μmol/kg TIW (6doses) (), and 2 μmol/kg TIW (6 doses) (▾) on the weight of Balb/c miceversus untreated controls (▪).

FIG. 6 shows the activity of Example 11 at 1 μmol/kg TIW for 2 weeks (6doses) on FR-positive KB tumors with (□) and without (▪) 40 μmol/kg EC20(rhenium complex) versus untreated controls (); Example 11 alone showed5/5 complete responses; Example 11+EC20 showed 0/5 complete responses.

FIG. 7 shows the absence of an effect by Example 11 at 1 μmol/kg TIW for2 weeks (6 doses) on the weight of nu/nu mice with (□) and without (▪)40 μmol/kg EC20 (rhenium complex) versus untreated controls ().

FIG. 8 shows the activity of Example 11 at 1 μmol/kg TIW for 2 weeks (6doses) on s.c. human xenograft KB tumors implanted in nude mice with (b)and without (c) 40 μmol/kg EC20 (rhenium complex) versus untreatedcontrols (a); Example 11 alone showed 5/5 complete responses; Example11+EC20 showed 0/5 complete responses.

FIG. 9 shows the absence of an effect by Example 11 at 1 μmol/kg TIW for2 weeks (6 doses) on the weight of nude mice with (b) and without (c) 40μmol/kg EC20 (rhenium complex) versus untreated controls (a).

FIG. 10 shows the activity of Example 11 at 2 μmol/kg TIW (e) on folatereceptor positive human tumors in nude mice as compared to a mixture ofthe unconjugated base drugs, mitomycin C and desacetylvinblastinemonohydrazide, at 0.5 μmol/kg TIW (b), 1 μmol/kg TIW (c), and 2 μmol/kgTIW (d), and compared to untreated controls (a).

FIG. 11 shows the absence of an effect by Example 11 at 2 μmol/kg TIWfor 2 weeks (e) on the weight of nude mice compared to controls (a).Weight loss occurred at the all three doses of the mixture of theunconjugated base drugs, mitomycin C and desacetylvinblastinemonohydrazide (0.5 μmol/kg TIW (b), 1 μmol/kg TIW (c), 2 μmol/kg TIW(d)). The high dose (d) was discontinued prior to day 20.

FIG. 12 shows the activity of Example 11 on three sizes of large KBtumors, 250 mm³ (b), 500 mm³ (c), and 750 mm³ (d) in nu/nu mice at 2μmol/kg TIW for 2 weeks compared to controls (a).

FIG. 13 shows the activity of Example 11 (e) compared to conjugates ofonly the single drug mitomycin C (b), desacetylvinblastine monohydrazide(c), or a mixture of those two single drug conjugates (d), compared tocontrols (a).

FIG. 14 shows the absence of activity of Example 11 (b) at 2 μmol/kg TIWfor two weeks of treatment on folate receptor negative 4T1 tumors inBablb/c mice, compared to controls (a). The data in FIG. 14 show thatExample 11 (b) does not have any effect on the tumors compared tocontrols (a) due to the absence of folate receptors on those tumors.

FIG. 15 shows the activity of Example 12 on ³H-thymidine incorporationinto FR-positive KB cells

DETAILED DESCRIPTION

Ligand conjugates of drugs, and analogs and derivatives thereof, aredescribed herein. The conjugates include cell receptor binding ligands,including ligands of cell surface receptors, that are covalentlyattached to two or more drugs that may be targeted to cells, includingpathogenic cells. The conjugates described herein may also include apolyvalent linker for attaching the ligands to the drugs.

Receptor binding drug delivery conjugates comprising a receptor bindingmoiety (B), a polyvalent linker (L), and two or more drugs, or druganalogs or drug derivatives, (D)_(n) are described, where n is greaterthan or equal to 2. In the delivery conjugates described herein, thereceptor binding moiety (B) and the two or more drugs (D)_(n) are eachbound to the polyvalent linker (L), through an independently selectedheteroatom linker (l_(H)). The polyvalent linker (L) comprises one ormore spacer linkers, heteroatom linkers, and releasable linkers, andcombinations thereof, in any order.

In one embodiment, a receptor binding drug delivery conjugate isdescribed. The drug delivery conjugate comprises a ligand, such as aligand of a cell surface receptor, two or more drugs, or analogs orderivatives thereof, and optionally a polyvalent linker, which may begenerally represented by the formula

(B)-(L)-(D)_(n)

wherein (B) represents a receptor binding moiety; (D) represents a drug,or analog or derivative thereof, to be targeted to a cell by thereceptor binding moiety; (L) represents a polyvalent linker, and n is aninteger greater than 1. The polyvalent linker (L) can comprise multiplelinkers covalently attached to each other. For example, the polyvalentlinker (L) can comprise one or more spacer linkers (l_(s)), and/orreleasable linkers (l_(r)), each connected to the other, and to theligand and the drug, by one or more heteroatom linkers (l_(H)). Thesevarious linkers may be selected and placed in any order to construct thepolyvalent linker (L).

Illustratively, the polyvalent linker (L) may be constructed from one ormore of the following bivalent linkers:

-(L)-

-(l_(r))_(c)-

-(l_(s))_(a-)

-(l_(s))_(a)-(l_(r))_(c)-

-(l_(r))_(c)-(l_(s))_(a)-

-(l_(H))_(b)-(l_(r))_(c)-

-(l_(r))_(c)-(l_(H))_(b)-

-(l_(H))_(d)-(l_(r))_(c)-(l_(H))_(e)-

-(l_(s))_(a)-(l_(H))_(b)-(l_(r))_(c)-

-(l_(r))_(c)-(l_(H))_(b)-(l_(s))_(a)-

-(l_(H))_(d)-(l_(s))_(a)-(l_(r))_(c)-(l_(H))_(e)-

-(l_(H))_(d)-(l_(r))_(c)-(l_(s))_(a)-(l_(H))_(e)-

-(l_(H))_(d)-(l_(s))_(a)-(l_(H))_(b)-(l_(r))_(c)-(l_(H))_(e)-

-(l_(H))_(d)-(l_(r))_(c)-(l_(H))_(b)-(l_(s))_(a)-(l_(H))_(e)-

-(l_(s))_(a)-(l_(r))_(c)-(l_(H))_(b)-

-[(l_(s))_(a)-(l_(H))_(b)]_(d)-(l_(r))_(c)-(l_(H))_(e)-

wherein a, b, c, d, and e are integers, such as integers in the rangefrom 0 to about 4, and (l_(s)), (l_(H)), and (l_(r)) are the spacerlinkers, releasable linkers, heteroatom linkers, respectively.Additional illustrative examples of bivalent linkers that may be used toconstruct the polyvalent linkers described herein are described in U.S.patent application Ser. No. 10/765,336 (also found as U.S. patentapplication publication no. US 2005/0002942 A1) and PCT internationalpublication no. WO2006/012527, the entirety of the disclosures of whichare incorporated herein by reference.

It is to be understood that the polyvalent linkers may connect thereceptor binding moiety to the two or more drugs in a variety ofstructural configurations, including but not limited to the followingillustrative general formulae:

where B is the receptor binding ligand, each of (L¹), (L²), and (L³) isa polyvalent linker constructed from one or more spacer, releasable,and/or heteroatom linkers, and each of (D¹), D², and D³ is a drug, or ananalog or derivative thereof. Other variations, including additionaldrugs, or analogs or derivatives thereof, additional linkers, andadditional configurations of the arrangement of each of (B), (L), and(D), are also contemplated herein.

In one variation, more than one receptor binding ligand is included inthe drug delivery conjugates described herein, including but not limitedto the following illustrative general formulae:

where each B is a receptor binding ligand, each of (L¹), (L²), and (L³)is a polyvalent linker constructed from one or more spacer, releasable,and/or heteroatom linkers, and each of (D¹), D², and D³ is a drug, or ananalog or derivative thereof. Other variations, including additionaldrugs, or analogs or derivatives thereof, additional linkers, andadditional configurations of the arrangement of each of (B), (L), and(D), are also contemplated herein. In one variation, the receptorbinding ligands are for the same receptor, and in another variation, thereceptor binding ligands are for different receptors. It is appreciated,and as shown in the above formulae, that more than one polyvalent linkermay be included in the drug delivery conjugates described herein. It isunderstood that in one aspect, the number of linkers are selecteddepending upon the configuration of the receptor binding ligands, andthe drugs.

For example, in one illustrative embodiment of the manner in whichlinkers are covalently assembled to form the polyvalent linker, or partof the polyvalent linker, heteroatom linkers, spacer linkers, andreleasable linkers are connected to form a polyvalent group of theformula:

where the formula may also be represented as

wherein (l_(s))¹ is the tripeptide Asp-Asp-Asp, (l_(s))² is Cys,(l_(r))¹ is S—S, (l_(s))³ is CH₂CH₂, (l_(H))¹ is O, (l_(r))² isC(O)NHNH, (l_(s))⁴ is w-Lys, (l_(s))⁵ is C(O)CH2CH2, (l_(r))³ is S—S,and (l_(s))⁶ is CH₂CH₂.

The ligands of cell surface receptors useful in the conjugates describedherein include, but are not limited to, vitamins, and other moietiesthat bind to a vitamin receptor, transporter, or other surface-presentedprotein that specifically binds vitamins, or analog or derivativethereof, peptide ligands identified from library screens, tumorcell-specific peptides, tumor cell-specific aptamers, tumorcell-specific carbohydrates, tumor cell-specific monoclonal orpolyclonal antibodies, Fab or scFv (i.e., a single chain variableregion) fragments of antibodies such as, for example, an Fab fragment ofan antibody directed to EphA2 or other proteins specifically expressedor uniquely accessible on metastatic cancer cells, small organicmolecules derived from combinatorial libraries, growth factors, such asEGF, FGF, insulin, and insulin-like growth factors, and homologouspolypeptides, somatostatin and its analogs, transferrin, lipoproteincomplexes, bile salts, selectins, steroid hormones, Arg-Gly-Aspcontaining peptides, retinoids, various Galectins, δ-opioid receptorligands, cholecystokinin A receptor ligands, ligands specific forangiotensin AT1 or AT2 receptors, peroxisome proliferator-activatedreceptor λ ligands, β-lactam antibiotics such as penicillin, smallorganic molecules including antimicrobial drugs, and other moleculesthat bind specifically to a receptor preferentially expressed on thesurface of tumor cells or on an infectious organism, antimicrobial andother drugs designed to fit into the binding pocket of a particularreceptor based on the crystal structure of the receptor or other cellsurface protein, ligands of tumor antigens or other moleculespreferentially expressed on the surface of tumor cells, or fragments ofany of these molecules. An example of a tumor-specific antigen thatcould function as a binding site for ligand-drug, or analog orderivative thereof, conjugates include extracellular epitopes of amember of the Ephrin family of proteins, such as EphA2. EphA2 expressionis restricted to cell-cell junctions in normal cells, but EphA2 isdistributed over the entire cell surface in metastatic tumor cells.Thus, EphA2 on metastatic cells would be accessible for binding to, forexample, an Fab fragment of an antibody conjugated to a drug, or analogor derivative thereof, whereas the protein would not be accessible forbinding to the Fab fragment on normal cells, resulting in a ligand-drugconjugate specific for metastatic cancer cells.

In one embodiment, the receptor binding moiety is a vitamin, or avitamin receptor binding analog or derivative thereof, such as vitaminsand analogs and derivatives thereof that are capable of binding vitaminreceptors.

The vitamins that can be used in accordance with the methods andcompounds described herein include carnitine, inositol, lipoic acid,pyridoxal, ascorbic acid, niacin, pantothenic acid, folic acid,riboflavin, thiamine, biotin, vitamin B₁₂, vitamins A, D, E and K, otherrelated vitamin molecules, analogs and derivatives thereof, andcombinations thereof. These vitamins, and their receptor-binding analogsand derivatives, constitute illustrative targeting entities that can becoupled with the drug compounds, or their analogs or derivatives, by thepolyvalent linkers (L) described herein to make drug deliveryconjugates.

In one illustrative aspect, the vitamin can be folic acid, a folic acidanalog, or another folate receptor-binding molecule. Exemplary ofanalogs of folate that can be used include folinic acid,pteroylpolyglutamic acid, pteroic acid and other amino acid derivativesthereof, and folate receptor-binding pteridines such astetrahydropterins, dihydrofolates, tetrahydrofolates, and their deazaand dideaza analogs. The terms “deaza” and “dideaza” analogs refers tothe art recognized analogs having a carbon atom substituted for one ortwo nitrogen atoms in the naturally occurring folic acid structure. Forexample, the deaza analogs include the 1-deaza, 3-deaza, 5-deaza,8-deaza, and 10-deaza analogs. The dideaza analogs include, for example,1,5 dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs. Theforegoing folic acid analogs are conventionally termed “folates,”reflecting their capacity to bind to folate receptors. Other folatereceptor-binding analogs include aminopterin, amethopterin(methotrexate), N¹⁰-methylfolate, 2-deamino-hydroxyfolate, deaza analogssuch as 1-deazamethopterin or 3-deazamethopterin, and3′,5′-dichloro-4-amino-4-deoxy-N¹⁰-methylpteroylglutamic acid(dichloromethotrexate). Other suitable ligands capable of binding tofolate receptors to initiate receptor mediated endocytotic transport ofthe drug delivery conjugate include antibodies to the folate receptor.Accordingly, in one illustrative aspect, a vinca compound in complexwith an antibody to a folate receptor can be used to triggertransmembrane transport of the complex.

Illustrative embodiments of vitamin analogs and/or derivatives alsoinclude analogs and derivatives of biotin such as biocytin, biotinsulfoxide, oxybiotin and other biotin receptor-binding compounds, andthe like. It is appreciated that analogs and derivatives of the othervitamins described herein are also contemplated herein.

Any shape of the described conjugates is contemplated herein, and isdetermined by the manner in which the drugs, receptor-binding moiety,and various polyvalent linkers are connected. In one aspect, the overallthree-dimensional shape of the conjugates described herein are linear.In another aspect, the overall three-dimensional shape of the conjugatesdescribed herein are “Y” or “T” shaped. In another aspect, the overallthree-dimensional shape of the conjugates described herein are “X”shaped or cross-shaped. In another

In one illustrative embodiment of the drug delivery conjugates describedherein, the polyvalent linker includes at least one releasable linker(l_(r)). In another illustrative embodiment of the drug deliveryconjugates described herein, the polyvalent linker includes at least tworeleasable linkers (l_(r))₂. In another illustrative aspect, thepolyvalent linker (L) includes at least one releasable linkers (l_(r))that is not a disulfide releasable linker. In another illustrativeaspect, the polyvalent linker (L) has at least two releasable linkers(l_(r))₂ where one releasable linker is not a disulfide releasablelinker. It is appreciated that when more than one releasable linker isincluded in the polyvalent linker, those releasable linkers may beadjacent. It is further appreciated that when two releasable linkers areadjacent in the polyvalent linker, the two releasable linkers maycooperate to cause release of the drug.

The term “releasable linker” as used herein, and also known as cleavablelinker, refers to a linker that includes at least one bond that can bebroken under physiological conditions (e.g., a pH-labile, acid-labile,oxidatively-labile, or enzyme-labile bond). It should be appreciatedthat such physiological conditions resulting in bond breaking includestandard chemical hydrolysis reactions that occur, for example, atphysiological pH, or as a result of compartmentalization into a cellularorganelle such as an endosome having a lower pH than cytosolic pH.

It is understood that a cleavable bond can connect two adjacent atomswithin the releasable linker and/or connect other linkers or (B) and/or(D), as described herein, at either or both ends of the releasablelinker. In the case where a cleavable bond connects two adjacent atomswithin the releasable linker, following breakage of the bond, thereleasable linker is broken into two or more fragments. Alternatively,in the case where a cleavable bond is between the releasable linker andanother moiety, such as an heteroatom linker, a spacer linker, anotherreleasable linker, the drug, or analog or derivative thereof, or thevitamin, or analog or derivative thereof, following breakage of thebond, the releasable linker is separated from the other moiety.

The lability of the cleavable bond can be adjusted by, for example,substitutional changes at or near the cleavable bond, such as includingalpha branching adjacent to a cleavable disulfide bond, increasing thehydrophobicity of substituents on silicon in a moiety having asilicon-oxygen bond that may be hydrolyzed, homologating alkoxy groupsthat form part of a ketal or acetal that may be hydrolyzed, and thelike.

Illustrative mechanisms for cleavage of the bivalant linkers describedherein include the following 1,4 and 1,6 fragmentation mechanisms

where X is an exogenous or endogenous nucleophile, glutathione, orbioreducing agent, and the like, and either of Z or Z′ is the vitamin,or analog or derivative thereof, or the drug, or analog or derivativethereof, or a vitamin or drug moiety in conjunction with other portionsof the polyvalent linker. It is to be understood that although the abovefragmentation mechanisms are depicted as concerted mechanisms, anynumber of discrete steps may take place to effect the ultimatefragmentation of the polyvalent linker to the final products shown. Forexample, it is appreciated that the bond cleavage may also occur byacid-catalyzed elimination of the carbamate moiety, which may beanchimerically assisted by the stabilization provided by either the arylgroup of the beta sulfur or disulfide illustrated in the above examples.In those variations of this embodiment, the releasable linker is thecarbamate moiety. Alternatively, the fragmentation may be initiated by anucleophilic attack on the disulfide group, causing cleavage to form athiolate. The thiolate may intermolecularly displace a carbonic acid orcarbamic acid moiety and form the corresponding thiacyclopropane. In thecase of the benzyl-containing polyvalent linkers, following anillustrative breaking of the disulfide bond, the resulting phenylthiolate may further fragment to release a carbonic acid or carbamicacid moiety by forming a resonance stabilized intermediate. In any ofthese cases, the releasable nature of the illustrative polyvalentlinkers described herein may be realized by whatever mechanism may berelevant to the chemical, metabolic, physiological, or biologicalconditions present.

Other illustrative mechanisms for bond cleavage of the releasable linkerinclude oxonium-assisted cleavage as follows:

where Z is the vitamin, or analog or derivative thereof, or the drug, oranalog or derivative thereof, or each is a vitamin or drug moiety inconjunction with other portions of the polyvalent linker, such as a drugor vitamin moiety including one or more spacer linkers, heteroatomlinkers, and/or other releasable linkers. In this embodiment,acid-catalyzed elimination of the carbamate leads to the release of CO₂and the nitrogen-containing moiety attached to Z, and the formation of abenzyl cation, which may be trapped by water, or any other Lewis base.

Another illustrative mechanism involves an arrangement of thereleasable, spacer, and heteroatom linkers in such a way that subsequentto the cleavage of a bond in the polyvalent linker, released functionalgroups chemically assist the breakage or cleavage of additional bonds,also termed anchimeric assisted cleavage or breakage. An illustrativeembodiment of such a polyvalent linker or portion thereof includescompounds having the formula:

where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is aninteger selected from 0, 1, 2, and 3, R is hydrogen, or a substituent,including a substituent capable of stabilizing a positive chargeinductively or by resonance on the aryl ring, such as alkoxy, and thelike, and either of Z or Z′ is the vitamin, or analog or, derivativethereof, or the drug, or analog or derivative thereof, or a vitamin ordrug moiety in conjunction with other portions of the polyvalent linker.It is appreciated that other substituents may be present on the arylring, the benzyl carbon, the carbamate nitrogen, the alkanoic acid, orthe methylene bridge, including but not limited to hydroxy, alkyl,alkoxy, alkylthio, halo, and the like. Assisted cleavage may includemechanisms involving benzylium intermediates, benzyne intermediates,lactone cyclization, oxonium intermediates, beta-elimination, and thelike. It is further appreciated that, in addition to fragmentationsubsequent to cleavage of the releasable linker, the initial cleavage ofthe releasable linker may be facilitated by an anchimerically assistedmechanism.

In this embodiment, the hydroxyalkanoic acid, which may cyclize,facilitates cleavage of the methylene bridge, by for example an oxoniumion, and facilitates bond cleavage or subsequent fragmentation afterbond cleavage of the releasable linker. Alternatively, acid catalyzedoxonium ion-assisted cleavage of the methylene bridge may begin acascade of fragmentation of this illustrative polyvalent linker, orfragment thereof. Alternatively, acid-catalyzed hydrolysis of thecarbamate may facilitate the beta elimination of the hydroxyalkanoicacid, which may cyclize, and facilitate cleavage of methylene bridge, byfor example an oxonium ion. It is appreciated that other chemicalmechanisms of bond breakage or cleavage under the metabolic,physiological, or cellular conditions described herein may initiate sucha cascade of fragmentation. It is appreciated that other chemicalmechanisms of bond breakage or cleavage under the metabolic,physiological, or cellular conditions described herein may initiate sucha cascade of fragmentation.

In one embodiment, the polyvalent linkers described herein are compoundsof the following formulae

where n is an integer selected from 1 to about 4; R^(a) and R^(b) areeach independently selected from the group consisting of hydrogen andalkyl, including lower alkyl such as C₁-C₄ alkyl that are optionallybranched; or R^(a) and R^(b) are taken together with the attached carbonatom to form a carbocyclic ring; R is an optionally substituted alkylgroup, an optionally substituted acyl group, or a suitably selectednitrogen protecting group; and (*) indicates points of attachment forthe drug, vitamin, imaging agent, diagnostic agent, other polyvalentlinkers, or other parts of the conjugate.

In another embodiment, the polyvalent linkers described herein includecompounds of the following formulae

where m is an integer selected from 1 to about 4; R is an optionallysubstituted alkyl group, an optionally substituted acyl group, or asuitably selected nitrogen protecting group; and (*) indicates points ofattachment for the drug, vitamin, imaging agent, diagnostic agent, otherpolyvalent linkers, or other parts of the conjugate.

In another embodiment, the polyvalent linkers described herein includecompounds of the following formulae

where m is an integer selected from 1 to about 4; R is an optionallysubstituted alkyl group, an optionally substituted acyl group, or asuitably selected nitrogen protecting group; and (*) indicates points ofattachment for the drug, vitamin, imaging agent, diagnostic agent, otherpolyvalent linkers, or other parts of the conjugate.

In another embodiment, the releasable, spacer, and heteroatom linkersmay be arranged in such a way that subsequent to the cleavage of a bondin the polyvalent linker, released functional groups chemically assistthe breakage or cleavage of additional bonds, also termed anchimericassisted cleavage or breakage. An illustrative embodiment of such apolyvalent linker or portion thereof includes compounds having theformula:

where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is aninteger selected from 0, 1, 2, and 3, R is hydrogen, or a substituent,including a substituent capable of stabilizing a positive chargeinductively or by resonance on the aryl ring, such as alkoxy, and thelike, and the symbol (*) indicates points of attachment for additionalspacer, heteroatom, or releasable linkers fowling the polyvalent linker,or alternatively for attachment of the drug, or analog or derivativethereof, or the vitamin, or analog or derivative thereof. It isappreciated that other substituents may be present on the aryl ring, thebenzyl carbon, the alkanoic acid, or the methylene bridge, including butnot limited to hydroxy, alkyl, alkoxy, alkylthio, halo, and the like.Assisted cleavage may include mechanisms involving benzyliumintermediates, benzyne intermediates, lactone cyclization, oxoniumintermediates, beta-elimination, and the like. It is further appreciatedthat, in addition to fragmentation subsequent to cleavage of thereleasable linker, the initial cleavage of the releasable linker may befacilitated by an anchimerically assisted mechanism.

In another embodiment, the polyvalent linker includes heteroatomlinkers, spacer linkers, and releasable linkers connected to form apolyvalent 3-thiosuccinimid-1-ylalkyloxymethyloxy group, illustrated bythe following formula

where n is an integer from 1 to 6, the alkyl group is optionallysubstituted, and the methyl is optionally substituted with an additionalalkyl or optionally substituted aryl group, each of which is representedby an independently selected group R. The (*) symbols indicate points ofattachment of the polyvalent linker fragment to other parts of theconjugates described herein.

In another embodiment, the polyvalent linker includes hetero atomlinkers, spacer linkers, and releasable linkers connected to form apolyvalent 3-thiosuccinimid-1-ylalkylcarbonyl group, illustrated by thefollowing formula

where n is an integer from 1 to 6, and the alkyl group is optionallysubstituted. The (*) symbols indicate points of attachment of thepolyvalent linker fragment to other parts of the conjugates describedherein. In another embodiment, the polyvalent linker includes heteroatomlinkers, spacer linkers, and releasable linkers connected to form apolyvalent 3-thioalkylsulfonylalkyl(disubstituted silyl)oxy group, wherethe disubstituted silyl is substituted with alkyl and/or optionallysubstituted aryl groups.

In another embodiment, the polyvalent linker includes heteroatomlinkers, spacer linkers, and releasable linkers connected to form apolyvalent dithioalkylcarbonylhydrazide group, or a polyvalent3-thiosuccinimid-1-ylalkylcarbonylhydrazide, illustrated by thefollowing formulae

where n is an integer from 1 to 6, the alkyl group is optionallysubstituted, and the hydrazide forms an hydrazone with (B), (D), oranother part of the polyvalent linker (L). The (*) symbols indicatepoints of attachment of the polyvalent linker fragment to other parts ofthe conjugates described herein.

In another embodiment, the polyvalent linker includes heteroatomlinkers, spacer linkers, and releasable linkers connected to form apolyvalent 3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene group,illustrated by the following formula

where each n is an independently selected integer from 1 to 6, eachalkyl group independently selected and is optionally substituted, suchas with alkyl or optionally substituted aryl, and where the alkylideneforms an hydrazone with (B), (D), or another part of the polyvalentlinker (L). The (*) symbols indicate points of attachment of thepolyvalent linker fragment to other parts of the conjugates describedherein.

In another embodiment, the polyvalent linker includes heteroatomlinkers, spacer linkers, and releasable linkers connected to form apolyvalent 3-thio or 3-dithioarylalkyloxycarbonyl group, 3-thio or3-dithioarylalkylaminocarbonyl group, a polyvalent 3-thio or3-dithioalkyloxycarbonyl, or a polyvalent 3-thio or3-dithioalkylaminocarbonyl, where the alkyl carbonyl forms a carbonate,a carbamate, or urea with (B), (D), or another part of the polyvalentlinker (L). Illustratively, the alkyl group is ethyl.

In another embodiment, the polyvalent linker includes heteroatomlinkers, spacer linkers, and releasable linkers connected to form apolyvalent 3-dithioalkylamino group, where the amino forms a vinylogousamide with (B), (D), or another part of the polyvalent linker (L).Illustratively, the alkyl group is ethyl.

In another embodiment, the polyvalent linker includes heteroatomlinkers, spacer linkers, and releasable linkers connected to form apolyvalent 1-alkoxycycloalkylenoxy group, a polyvalentalkyleneaminocarbonyl(dicarboxylarylene)carboxylate group, a polyvalent3-dithioalkyloxycarbonyl group, a polyvalent3-dithioalkyloxycarbonylhydrazide group, a polyvalent.

In another embodiment, the polyvalent linker includes at least onespacer linker that is a peptide formed from amino acids. In one aspect,the peptide includes naturally occurring amino acids, and stereoisomersthereof. In another aspect, the peptide is formed only from naturallyoccurring amino acids, and stereoisomers thereof.

Additional illustrative examples of spacer and releasable linkers areshown in Table 1 and 2, where the (*) indicates the point of attachmentto another linker, to the vinca alkaloid, or analog or derivativethereof, or to the receptor binding moiety.

TABLE 1 Contemplated spacer and heteroatom linkers, and combinationsthereof.

TABLE 2 Contemplated releasable and heteroatom linkers, and combinationsthereof.

Any variety of drugs may be included in the drug delivery conjugatesdescribed herein. In one illustrative embodiment, the drugs are selectedbased on activity against one or more populations of pathogenic cells.In one aspect, those pathogenic cells are cancer cells, including solidtumors.

In another illustrative embodiment, the drugs are selected based onactivity against one or more populations of pathogenic cells with aparticular mechanism of action. Illustrative mechanisms of actioninclude alkylating agents, microtubule inhibitors, including those thatstabilize and/or destabilize microtubule formation, includingbeta-tubulin agents, cyclin dependent kinase (CDK) inhibitors,topoisomerase inhibitors, protein synthesis inhibitors, protein kinaseinhibitors, including Ras, Raf, PKC, PI3K, and like inhibitors,transcription inhibitor, antifolates, heat shock protein blockers, andthe like.

Illustrative alkylating agents include, but are not limited to,mitomycins CBI, and the like. Illustrative cyclin dependent kinase (CDK)inhibitors include, but are not limited to, CYC202, seliciclib,R-roscovitine, AGM-1470, and the like. Illustrative topoisomeraseinhibitors include, but are not limited to, doxorubicin, otheranthracyclines, and the like. Illustrative protein synthesis inhibitorsinclude, but are not limited to, bruceantin, and the like. Illustrativeprotein kinase inhibitors, including Ras, Raf, PKC, PI3K, and likeinhibitors, include but are not limited to L-779,450, R115777, and thelike. Illustrative transcription inhibitors include, but are not limitedto, α-amanatin, actinomycin, and the like. Illustrative antifolatesinclude, but are not limited to, methotrexate, and the like.Illustrative heat shock protein blockers include, but are not limitedto, geldanamycin, and the like.

Illustrative microtubule inhibitors, including those that stabilizeand/or destabilize microtubule formation, including β-tubulin agents,microtubule poisons, and the like. Illustrative microtubule poisons thatbind to selected receptors include, but are not limited to, inhibitorsbiding to the vinca binding site such as arenastatin, dolastatin,halichondrin B, maytansine, phomopsin A, rhizoxin, ustiloxin,vinblastine, vincristine, and the like, stabilizers binding to the taxolbinding site such as discodermalide, epothilone, taxol, paclitaxol, andthe like, inhibitors binding to the colchicine binding site such as,colchicine, combretastatin, curacin A, podophyllotoxin, steganacine, andthe like, and others binding to undefined sites such as cryptophycin,tubulysins, and the like.

In one embodiment of the drug delivery conjugates described herein, atleast one of the drugs is a microtubule inhibitor, or an analog orderivative thereof. In another embodiment, at least one of the drugs isa DNA alkylation agent. In another embodiment, at least one of the drugsis a DNA alkylation agent, and at least one other of the drugs is amicrotubule inhibitor. alklaloids described herein include all membersof the vinca indole-dihydroindole family of alkaloids, such as but notlimited to vindesine, vinblastine, vincristine, catharanthine,vindoline, leurosine, vinorelbine, imidocarb, sibutramine, toltrazuril,vinblastinoic acid, and the like, and analogs and derivatives thereof.

In another embodiment of the drug delivery conjugates described herein,at least one of the drugs is a P-glycoprotein (PGP) inhibitor. Inanother embodiment, at least one of the drugs included on the drugdelivery conjugates described herein is a PGP inhibitor, and at leastone other of the drugs included on the drug delivery conjugates is a PGPsubstrate. Illustratively in this latter embodiment, the PGP substrateis a DNA alkylating agent. Referring to this embodiment, it isappreciated that pairing a PGP inhibitor with a PGP substrate, such as aDNA alkylating agent including, but not limited to, any of themitomycins like mitomycin C, mitomycin A, and the like may improve theoverall performance of the drug that is otherwise a PGP substrate. Inthe releasable conjugates described herein, the PGP inhibitor drug andthe PGP substrate drug are both released in the cell after endocytosis.In that manner, the PGP inhibitor drug may improve the overall efficacyand/or potency of the PGP substrate drug. In addition, the PGP inhibitormay reduces PGP expression, which in turn will decrease efflux of one ormore of the drugs included on the multidrug conjugates described hereinfrom the pathogenic cell. It is appreciated that the mitomycins, oranalogs or derivatives thereof, such as mitomycin C may operate as a PGPinhibitor, or down-regulator of PGP. It is further appreciated that thevinca alkaloid, or analog or derivative thereof, such as vinblastineanalogs and derivatives, may be a PGP substrate that is protected fromefflux from the pathogenic cell by the PGP inhibitor or down-regulator.

In another embodiment of the drug delivery conjugates described herein,at least one of the drugs is a vinca alkaloid, or an analog orderivative thereof. Vinca alklaloids described herein include allmembers of the vinca indole-dihydroindole family of alkaloids, such asbut not limited to vindesine, vinblastine, vincristine, catharanthine,vindoline, leurosine, vinorelbine, imidocarb, sibutramine, toltrazuril,vinblastinoic acid, and the like, and analogs and derivatives thereof.

As referred to herein, the vinca drugs useable in the conjugatesdescribed herein include all members of the vinca indole-dihydroindolefamily of alkaloids, such as vindesine, vinblastine, vincristine,catharanthine, vindoline, leurosine, vinorelbine, imidocarb,sibutramine, toltrazuril, vinblastinoic acid, and the like, and analogsand derivatives thereof. Illustratively, such analogs and derivativesinclude the 3-carboxazides described in U.S. Pat. No. 4,203,898; theN²-alkyl and other derivatives of4-desacetylvinblastine-3-carboxhydrazide described in U.S. Pat. No.4,166,810; leurosine hydrazide described in Neuss et al. TetrahedronLett. 783 (1968); the hydrazide derivatives described in Barnett et al.J. Med. Chem. 21:88 (1978); the C-4 ester derivatives described in U.S.Pat. Nos. 3,392,173 and 3,387,001; the dicarboxylic acid derivativesresulting from oxidation described in Langone et al. Anal. Biochem.95:214 (1979); and the vinca hydrazides described in EP 0 247 792 A2.Each of the foregoing patents and publications is incorporated herein byreference for all that it discloses regarding synthetic routes, andreaction conditions for preparing vinca compounds.

In one illustrative embodiment, the vinca drugs are compounds of theformula

wherein:

one of R¹ and R² is H, and the other is ethyl, and R³ is H, or R¹ isethyl R², and R³ are taken together to form —O—;

R⁴, R⁷, and R⁸ are each independently selected from H, alkyl, and acyl

R⁵ and R⁶ are each independently selected alkyl;

R⁹ is a group —NHNHR, where R is H, alkyl, or acyl;

R¹⁰ is H or acyl; and

R¹¹ is ethyl.

In one aspect, the vinca drugs are compounds of the above formulawherein R⁴ and R⁸ are each H; and R⁵, R⁶, R⁹, and R¹⁰ are each methyl.

In another embodiment, a receptor binding drug delivery conjugate isdescribed comprising a receptor binding moiety, a polyvalent linker (L),a vinca alkaloid drug, or analog or derivative thereof, and anotherdrug, or analog or derivative thereof, wherein the receptor bindingmoiety, the vinca alkaloid, and the other drug are each bound to thepolyvalent linker (L), through an heteroatom linker (I_(H)). Thepolyvalent linker (L) comprises one or more spacer linkers, heteroatomlinkers, and releasable linkers, and combinations thereof, in any order.

In another embodiment, at least one of the drugs included on the drugdelivery conjugates described herein is an aclamycin, or an analog orderivative thereof. It may be that the aclamycins and analogs andderivatives thereof are PGP efflux pump substrates. In one aspect, atleast one other of the drugs included on the drug delivery conjugatesdescribed herein is an DNA alkylating agent, such as a mitomycin or ananalog or derivative thereof.

In another embodiment, at least one of the drugs included on the drugdelivery conjugates described herein is a DNA synthesis inhibitor, or ananalog or derivative thereof. In another embodiment, at least one of thedrugs included on the drug delivery conjugates described herein is aspindle formation inhibitor, or an analog or derivative thereof. In oneaspect, at least one of the drugs included on the drug deliveryconjugates described herein is a DNA synthesis inhibitor, or an analogor derivative thereof, and at least one other of the drugs included onthe drug delivery conjugates described herein is a spindle formationinhibitor, or an analog or derivative thereof.

In another embodiment, at least one of the drugs included on the drugdelivery conjugates described herein is a microtubule stabilizing agent,or an analog or derivative thereof. In another embodiment, at least oneof the drugs included on the drug delivery conjugates described hereinis a microtubule synthesis inhibitor, or an analog or derivativethereof. In another embodiment, at least one of the drugs included onthe drug delivery conjugates described herein is a microtubuledestabilizing agent, or an analog or derivative thereof.

In another embodiment, at least one of the drugs included on the drugdelivery conjugates described herein is a apoptosis inducing agent, oran analog or derivative thereof. In another embodiment, at least one ofthe drugs included on the drug delivery conjugates described herein is ataxol, or an analog or derivative thereof. In another embodiment, atleast one of the drugs included on the drug delivery conjugatesdescribed herein is an antifolate, or an analog or derivative thereof.In another embodiment, at least one of the drugs included on the drugdelivery conjugates described herein is a methotrexate, or an analog orderivative thereof. In one aspect, at least one of the drugs included onthe drug delivery conjugates described herein is an antifolate, or ananalog or derivative thereof, such as methotrexate, and at least oneother of the drugs included on the drug delivery conjugates describedherein is a taxol, or an analog or derivative thereof.

In another embodiment, at least one of the drugs included on the drugdelivery conjugates described herein is a folate, or an analog orderivative thereof. In another embodiment, at least one of the drugsincluded on the drug delivery conjugates described herein is a humanepidermal growth factor receptor-2 (HER-2) inhibitor, or an analog orderivative thereof. In another embodiment, at least one of the drugsincluded on the drug delivery conjugates described herein is aradiolabeled chemotherapy agent, such as cisplatin, and the like. In oneaspect, at least one of the drugs included on the drug deliveryconjugates described herein is an antifolate, or an analog or derivativethereof, such as methotrexate, and at least one other of the drugsincluded on the drug delivery conjugates described herein is a folate,or an analog or derivative thereof. In another aspect, at least one ofthe drugs included on the drug delivery conjugates described herein is ataxol, or an analog or derivative thereof; and at least one other of thedrugs included on the drug delivery conjugates described herein is aHER-2 inhibitor, or an analog or derivative thereof. In another aspect,at least one of the drugs included on the drug delivery conjugatesdescribed herein is a taxol, or an analog or derivative thereof, atleast one other of the drugs included on the drug delivery conjugatesdescribed herein is a radiolabeled chemotherapy agent, such ascisplatin, and at least one other of the drugs included on the drugdelivery conjugates described herein is a HER-2 inhibitor, or an analogor derivative thereof.

The drug delivery conjugates described herein can be prepared byconventional synthetic methods. The synthetic methods are chosendepending upon the selection of the heteroatom linkers, and thefunctional groups present on the spacer linkers and the releasablelinkers. In general, the relevant bond forming reactions are describedin Richard C. Larock, “Comprehensive Organic Transformations, a guide tofunctional group preparations,” VCH Publishers, Inc. New York (1989),and in Theodora E. Greene & Peter G. M. Wuts, “Protective Groups ionOrganic Synthesis,” 2d edition, John Wiley & Sons, Inc. New York (1991),the disclosures of which in their entirety are incorporated herein byreference. Additional synthetic routes and reaction conditions aredescribed in U.S. patent application publication no. US 2005/0002942 A1.

Illustratively, the drug delivery conjugates described herein may beprepared using both linear and convergent synthetic routes. Illustrativeintermediates useable in such routes include intermediates comprising apolyvalent linker that includes a coupling group at each end suitablefor covalent attachment to the receptor binding moiety, or analog orderivative thereof, and the vinca alkaloid, or analog or derivativethereof. Other illustrative intermediates useable in such routes includeintermediates comprising a receptor binding moiety, or analog orderivative thereof, attached to a polyvalent linker, which includes acoupling group. Other illustrative intermediates useable in such routesinclude intermediates comprising a vinca alkaloid, or analog orderivative thereof, attached to a polyvalent linker, which includes acoupling group. In either case, the coupling group may be a nucleophile,an electrophile, or a precursor thereof.

In one illustrative embodiment synthetic intermediates, the couplinggroup is a Michael acceptor, and the polyvalent linker includes areleasable linker having the formula —C(O)NHN═, —NHC(O)NHN═, or—CH₂C(O)NHN═. In one illustrative aspect, the coupling group and thepolyvalent linker are taken together to form a compound having theformula:

or a protected derivative thereof, where (D) is the vinca alkaloid, oran analog or a derivative thereof, capable of forming a hydrazone asillustrated herein; and n is an integer such as 1, 2, 3, or 4. Inanother illustrative aspect of the receptor binding drug deliveryconjugate intermediate described herein, a second linker is covalentlyattached to the above formula through an alkylthiol nucleophile includedon the second linker. In another illustrative aspect, the receptorbinding moiety, or analog or derivative thereof, is covalently attachedto the above formula through an alkylthiol nucleophile included on thatmoiety.

In another illustrative embodiment, the coupling group is a heteroatom,such as nitrogen, oxygen, or sulfur, and the polyvalent linker includesone or more heteroatom linkers and one or more spacer linkers covalentlyconnecting the receptor binding moiety to the coupling group. In oneillustrative aspect, the intermediate described herein includes acompound having the formula:

or a protected derivative thereof, where X is oxygen, nitrogen, orsulfur, and in is an integer such as 1, 2, or 3, and where (B), l_(s),and l_(H) are as defined herein. In one illustrative aspect, l_(H) is—NH—, and m is 1. In another illustrative aspect, l_(H) is —NH—, in is1, and X is —S—.

In another illustrative embodiment, the intermediate described hereinincludes a compound having the formula:

or a protected derivative thereof, where Y is H or a substituent,illustratively an electron withdrawing substituent, including but notlimited to nitro, cyano, halo, alkylsulfonyl, a carboxylic acidderivative, and the like, and where (B) and l_(s) are as defined herein.

In another illustrative embodiment of the intermediate described herein,the coupling group is a Michael acceptor, and the polyvalent linkerincludes one or more heteroatom linkers and one or more spacer linkerscovalently connecting the receptor binding moiety to the coupling group.In one illustrative aspect, the coupling group and the polyvalent linkerare taken together to form a compound having the formula:

or a protected derivative thereof, where X is oxygen, nitrogen, orsulfur, and m and n are independently selected integers, such as 1, 2,or 3, and where (B), l_(s), and l_(H) are as defined herein. In anotherillustrative aspect, the vinca alkaloid, or analog or derivativethereof, is covalently attached to the above formula through analkylthiol nucleophile included on the vinca alkaloid.

In another illustrative aspect, the intermediate includes compoundshaving the formulae:

or protected derivatives thereof, where AA is one or more amino acids,illustratively selected from the naturally occurring amino acids, orstereoisomers thereof, X is nitrogen, oxygen, or sulfur, Y is hydrogenor a substituent, illustratively an electron withdrawing substituent,including but not limited to nitro, cyano, halo, alkylsulfonyl, acarboxylic acid derivative, and the like, n and m are independentlyselected integers, such as 1, 2, or 3, and p is an integer such as 1, 2,3, 4, or 5.

AA can also be any other amino acid, such as any amino acid having thegeneral formula:

—N(R)—(CR′R″)_(t)—C(O)—

where R is hydrogen, alkyl, acyl, or a suitable nitrogen protectinggroup, R′ and R″ are hydrogen or a substituent, each of which isindependently selected in each occurrence, and t is an integer such as1, 2, 3, 4, or 5. Illustratively, R′ and/or R″ independently correspondto, but are not limited to, hydrogen or the side chains present onnaturally occurring amino acids, such as methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, andderivatives and protected derivatives thereof. The above describedformula includes all stereoisomeric variations. For example, the aminoacid may be selected from asparagine, aspartic acid, cysteine, glutamicacid, lysine, glutamine, arginine, serine, ornitine, threonine, and thelike. In another illustrative aspect of the vitamin receptor bindingdrug delivery conjugate intermediate described herein, the drug, or ananalog or a derivative thereof, includes an alkylthiol nucleophile.

Each of the above intermediates may be prepared using conventionalsynthetic routes. Additional synthetic routes and reaction conditionsare described in U.S. patent application publication no. US 2005/0002942A1 and PCT international publication no. WO 2006/012527.

The foregoing illustrative embodiments are intended to be illustrativeof the invention described herein, and should not be interpreted orconstrued as limiting in any way the invention as described herein. Forexample, compounds generally represented by the following illustrativevitamin-drug conjugate intermediate are to be included in the inventionas described herein

where R¹ and R² are each independently hydrogen or alkyl, such asmethyl; and l_(H) is a heteroatom, such as oxygen, sulfur, optionallysubstituted nitrogen, or optionally protected nitrogen, and the like.Two or more drugs, and optionally additional receptor-binding ligands,such as folates and analogs and derivatives thereof, may be covalentlyattached to this illustrative intermediate at (l_(H)), or at otherfunctional groups present, such as the amide nitrogen or carbonyl, theacid carboxylate, or the guanidine amino group.

In another embodiment, a folate ligand intermediate is described havingthe following formula

wherein m, n, and q are integers that are independently selected fromthe range of 0 to about 8; AA is an amino acid, R¹ is hydrogen, alkyl,or a nitrogen protecting group, and drugs are optionally attached at the(*) atoms. In one aspect, AA is a naturally occurring amino acid ofeither the natural or unnatural configuration. In another aspect, one ormore of AA in the fragment (—NH-AA-C(O)—)_(n) is a hydrophilic aminoacid. In another aspect, one or more of AA in the fragment(—NH-AA-C(O)—)_(n) is Asp and/or Arg. In another aspect, the integer ois 1 or greater. In another aspect, the integer m is 2 or greater. Thedrugs, or analogs or derivatives thereof, and optionally additionallinkers and additional receptor-binding ligands may be connected to theabove formula at the free NH side chains of the 2,ω-diaminoalkanoic acidfragments, or at the terminal carboxylate as indicated by the freevalences therein.

In another embodiment, a folate ligand intermediate is described havingthe following formula

wherein m, n, q, and p are integers that are independently selected fromthe range of 0 to about 8; AA is an amino acid, R¹ is hydrogen, alkyl,or a nitrogen protecting group, and drugs are optionally attached at the(*) atoms. In one aspect, AA is as a naturally occurring amino acid ofeither the natural or unnatural configuration. In another aspect, one ormore of AA in the fragment (—NH-AA-C(O)—)_(n) is a hydrophilic aminoacid. In another aspect, one or more of AA in the fragment(—NH-AA-C(O)—)_(n) is Asp and/or Arg. In another aspect, the integers oand p are 1 or greater. In another aspect, the integer m is 2 orgreater. The drugs, or analogs or derivatives thereof, and optionallyadditional linkers and additional receptor-binding ligands may beconnected to the above formula at the free NH side chains of the2,ω-diaminoalkanoic acid fragments, at the cyteinyl thiol groups, or atthe terminal carboxylate, as indicated by the free valences therein.

In another embodiment, a folate ligand intermediate is described havingthe following formula

wherein m, n, q, p, and r are integers that are independently selectedfrom the range of 0 to about 8; AA is an amino acid, R¹ is hydrogen,alkyl, or a nitrogen protecting group, and drugs are optionally attachedat the (*) atoms. In one aspect, AA is as a naturally occurring aminoacid of either the natural or unnatural configuration. In anotheraspect, one or more of AA in the fragment (—NH-AA-C(O)—)_(n) is ahydrophilic amino acid. In another aspect, one or more of AA in thefragment (—NH-AA-C(O)—)_(n) is Asp and/or Arg. In another aspect, theintegers o, p, and r are 1 or greater. In another aspect, the integer mis 2 or greater. The drugs, or analogs or derivatives thereof, andoptionally additional linkers and additional receptor-binding ligandsmay be connected to the above formula at the free NH side chains of the2,ω-diaminoalkanoic acid fragments, at the cyteinyl thiol groups, at theserinyl hydroxy groups, or at the terminal carboxylate, as indicated bythe free valences therein.

In another embodiment, a folate ligand intermediate that includesmitomycin as one of the drugs is described and having the followingformula

wherein m, n, and q are integers that are independently selected fromthe range of 0 to about 8; and AA is an amino acid. In one aspect, AA isas a naturally occurring amino acid of either the natural or unnaturalconfiguration. In another aspect, one or more of AA in the fragment(—NH-AA-C(O)—)_(n) is a hydrophilic amino acid. In another aspect, oneor more of AA in the fragment (—NH-AA-C(O)—)_(n) is Asp and/or Arg. Inanother aspect, the integer o is 1 or greater. In another aspect, theinteger m is 2 or greater. The drugs, or analogs or derivatives thereof,and optionally additional linkers and additional receptor-bindingligands may be connected to the above formula at the additional free NHside chains of the 2,ω-diaminoalkanoic acid fragments, or at theterminal carboxylate, as indicated by the free valences therein.

In another embodiment, a folate ligand multidrug conjugate that includesa mitomycin and a vinca alkaloid is described and having the followingformula

In another embodiment, a folate ligand multidrug conjugate that includesa mitomycin, an aclamycin, and a vinca alkaloid is described and havingthe following formula

In another embodiment, a pharmaceutical composition is described. Thepharmaceutical composition comprises a drug delivery conjugate describedherein in combination with a pharmaceutically acceptable carrier,excipient, and/or diluent therefor.

In another embodiment, a method for eliminating a population ofpathogenic cells in a host animal harboring the population of pathogeniccells is described. In one illustrative aspect, the members of thepathogenic cell population have an accessible binding site for areceptor binding moiety, or the analog or derivative thereof, and thatbinding site is uniquely expressed, overexpressed, or preferentiallyexpressed by the pathogenic cells. The method includes the step ofadministering to the host a drug delivery conjugate described herein, ora pharmaceutical composition thereof, as described herein.

Populations of pathogenic cells that may be treated using the methodsdescribed herein include, but at not limited to cancers, such asepithelial cancers of the ovary, mammary gland, colon, lung, nose,throat, brain, and other tumor cell types, infectious agents, activatedmacrophages, activated monocytes, and the like.

The drug delivery conjugates described herein can be used for both humanclinical medicine and veterinary applications. Thus, the host animalharboring the population of pathogenic cells and treated with the drugdelivery conjugates can be human or, in the case of veterinaryapplications, can be a laboratory, agricultural, domestic, or wildanimal. The drug delivery conjugates described herein can beadministered to host animals including, but not limited to, humans,laboratory animals such rodents (e.g., mice, rats, hamsters, etc.),rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats, andrabbits, agricultural animals such as cows, horses, pigs, sheep, goats,and wild animals in captivity such as bears, pandas, lions, tigers,leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.

The drug delivery conjugates described herein can be used to treat avariety of pathologies and pathogenic cells in host animals. As usedherein, “pathogenic cells” means cancer cells, infectious agents such asbacteria and viruses, bacteria- or virus-infected cells, activatedmacrophages capable of causing a disease state, and any other type ofpathogenic cells that uniquely express, preferentially express, oroverexpress ligand receptors, such as vitamin receptors or receptorsthat bind analogs or derivatives of vitamins. Pathogenic cells can alsoinclude any cells causing a disease state for which treatment with thedrug delivery conjugates results in reduction of the symptoms of thedisease. The pathogenic cells can also be host cells that are pathogenicunder some circumstances, such as cells of the immune system that areresponsible for graft versus host disease', but not pathogenic underother circumstances.

Thus, the population of pathogenic cells can be a cancer cell populationthat is tumorigenic, including benign tumors and malignant tumors, or itcan be non-tumorigenic. The cancer cell population can arisespontaneously or by such processes as mutations present in the germlineof the host animal or somatic mutations, or it can be chemically-,virally-, or radiation-induced. The invention can be utilized to treatsuch cancers as carcinomas, sarcomas, lymphomas, Hodgekin's disease,melanomas, mesotheliomas, Burkitt's lymphoma, nasopharyngeal carcinomas,leukemias, and myelomas. The cancer cell population can include, but isnot limited to, oral, thyroid, endocrine, skin, gastric, esophageal,laryngeal, pancreatic, colon, bladder, bone, ovarian, cervical, uterine,breast, testicular, prostate, rectal, kidney, liver, and lung cancers.

In embodiments where the pathogenic cell population is a cancer cellpopulation, the effect of drug delivery conjugate administration is atherapeutic response measured by reduction or elimination of tumor massor of inhibition of tumor cell proliferation. In the case of a tumor,the elimination can be an elimination of cells of the primary tumor orof cells that have metastasized or are in the process of dissociatingfrom the primary tumor. A prophylactic treatment with the drug deliveryconjugate to prevent return of a tumor after its removal by anytherapeutic approach including surgical removal of the tumor, radiationtherapy, chemotherapy, or biological therapy is also contemplated. Theprophylactic treatment can be an initial treatment with the drugdelivery conjugate, such as treatment in a multiple dose daily regimen,and/or can be an additional treatment or series of treatments after aninterval of days or months following the initial treatment(s).Accordingly, elimination of any of the pathogenic cell populationsdescribed above includes reduction in the number of pathogenic cells,inhibition of proliferation of pathogenic cells, a prophylactictreatment that prevents return of pathogenic cells, or a treatment ofpathogenic cells that results in reduction of the symptoms of disease.

In cases where cancer cells are being eliminated, the method describedherein can be used in combination with surgical removal of a tumor,radiation therapy, chemotherapy, or biological therapies such as otherimmunotherapies including, but not limited to, monoclonal antibodytherapy, treatment with immunomodulatory agents, adoptive transfer ofimmune effector cells, treatment with hematopoietic growth factors,cytokines and vaccination.

The method described herein is also applicable to populations ofpathogenic cells that cause a variety of infectious diseases. Forexample, the present invention is applicable to such populations ofpathogenic cells as bacteria, fungi, including yeasts, viruses,virus-infected cells, mycoplasma, and parasites. Infectious organismsthat can be treated with the drug delivery conjugates described hereinare any art-recognized infectious organisms that cause pathogenesis inan animal, including such organisms as bacteria that are gram-negativeor gram-positive cocci or bacilli. For example, Proteus species,Klebsiella species, Providencia species, Yersinia species, Erwiniaspecies, Enterobacter species, Salmonella species, Serratia species,Aerobacter species, Escherichia species, Pseudomonas species, Shigellaspecies, Vibrio species, Aeromonas species, Campylobacter species,Streptococcus species, Staphylococcus species, Lactobacillus species,Micrococcus species, Moraxella species, Bacillus species, Clostridiumspecies, Corynebacterium species, Eberthella species, Micrococcusspecies, Mycobacterium species, Neisseria species, Haemophilus species,Bacteroides species, Listeria species, Erysipelothrix species,Acinetobacter species, Brucella species, Pasteurella species, Vibriospecies, Flavobacterium species, Fusobacterium species, Streptobacillusspecies, Calymmatobacterium species, Legionella species, Treponemaspecies, Borrelia species, Leptospira species, Actinomyces species,Nocardia species, Rickettsia species, and any other bacterial speciesthat causes disease in a host animal can be treated with the drugdelivery conjugates described herein.

Of particular interest are bacteria that are resistant to antibioticssuch as antibiotic-resistant Streptococcus species and Staphlococcusspecies, or bacteria that are susceptible to antibiotics, but causerecurrent infections treated with antibiotics so that resistantorganisms eventually develop. Bacteria that are susceptible toantibiotics, but cause recurrent infections treated with antibiotics sothat resistant organisms eventually develop, can be treated with thedrug delivery conjugates described herein in the absence of antibiotics,or in combination with lower doses of antibiotics than would normally beadministered to a host animal, to avoid the development of theseantibiotic-resistant bacterial strains.

Diseases caused by viruses, such as DNA and RNA viruses, can also betreated with the drug delivery conjugates described herein. Such virusesinclude, but are not limited to, DNA viruses such as papilloma viruses,parvoviruses, adenoviruses, herpesviruses and vaccinia viruses, and RNAviruses, such as arenaviruses, coronaviruses, rhinoviruses, respiratorysyncytial viruses, influenza viruses, picornaviruses, paramyxoviruses,reoviruses, retroviruses, lentiviruses, and rhabdoviruses.

The drug delivery conjugates described herein can also be used to treatdiseases caused by any fungi, including yeasts, mycoplasma species,parasites, or other infectious organisms that cause disease in animals.Examples of fungi that can be treated with the method and drug deliveryconjugates described herein include fungi that grow as molds or areyeastlike, including, for example, fungi that cause diseases such asringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis,sporotrichosis, coccidioidomycosis, paracoccidio-idomycosis,muconnycosis, chromoblastomycosis, dermatophytosis, protothecosis,fusariosis, pityriasis, mycetoma, paracoccidioidomycosis,phaeohyphomycosis, pseudallescheriasis, sporotrichosis, trichosporosis,pneumocystis infection, and candidiasis.

The drug delivery conjugates described herein can also be used to treatparasitic infections including, but not limited to, infections caused bytapeworms, such as Taenia, Hymenolepsis, Diphyllobothrium, andEchinococcus species, flukes, such as Fasciolopsis, Heterophyes,Metagonimus, Clonorchis, Fasciola, Paragonimus, and Schitosoma species,roundworms, such as Enterobius, Trichuris, Ascaris, Ancylostoma,Necator, Strongyloides, Trichinella, Wuchereria, Brugia, Loa Onchocerca,and Dracunculus species, ameba, such as Naegleria and Acanthamoebaspecies, and protozoans, such as Plasmodium, Trypanosoma, Leishmania,Toxoplasma, Entamoeba, Giardia, Isospora, Cryptosporidium, andEnterocytozoon species.

The pathogenic cells to which the drug delivery conjugates are directedcan also be cells harboring endogenous pathogens, such as virus-,mycoplasma-, parasite- or bacteria-infected cells, if these cellspreferentially express ligand receptors, such as receptors for vitamins,or analogs or derivatives thereof.

In one embodiment, the drug delivery conjugates can be internalized intothe targeted pathogenic cells upon binding of the ligand to a receptor,transporter, or other surface-presented protein that specifically bindsthe ligand and which is preferentially expressed on the pathogeniccells. Such internalization can occur, for example, throughreceptor-mediated endocytosis. If the drug delivery conjugate contains areleasable linker, the ligand and the vinca compound can dissociateintracellularly and the vinca can act on its intracellular target.

In another illustrative embodiment, the ligand of the drug deliveryconjugate can bind to the pathogenic cell placing the vinca compound inclose association with the surface of the pathogenic cell. The vincacompound can then be released by cleavage of the releasable linker. Forexample, the vinca compound can be released by a protein disulfideisomerase if the releasable linker is a disulfide group. The vincacompound can then be taken up by the pathogenic cell to which thereceptor binding drug delivery conjugate is bound, or the vinca compoundcan be taken up by another pathogenic cell in close proximity thereto.Alternatively, the vinca compound could be released by a proteindisulfide isomerase inside the cell where the releasable linker is adisulfide group. The vinca compound may also be released by a hydrolyticmechanism, such as acid-catalyzed hydrolysis, as described above forcertain beta elimination mechanisms, or by an anchimerically assistedcleavage through an oxonium ion or lactonium ion producing mechanism.The selection of the releasable linker or linkers will dictate themechanism by which the vinca compound is released from the conjugate. Itis appreciated that such a selection can be pre-defined by theconditions under which the drug delivery conjugate will be used.

In another illustrative embodiment, where the linker does not comprise areleasable linker, the ligand moiety of the drug delivery conjugate canbind to the pathogenic cell placing the vinca compound on the surface ofthe pathogenic cell to target the pathogenic cell for attack by othermolecules capable of binding to the vinca compound. Alternatively, inthis embodiment, the drug delivery conjugates can be internalized intothe targeted cells upon binding, and the ligand moiety and the vincacompound can remain associated intracellularly with the vinca compoundexhibiting its effects without dissociation from the ligand moiety.

In still another embodiment, or in combination with the above-describedembodiments, where the drug delivery conjugate binds a vitamin receptoror another ligand receptor, the conjugate can bind to soluble vitaminreceptors present in the serum or to serum proteins, such as albumin,resulting in prolonged circulation of the conjugates relative to theunconjugated vinca compound, and in increased activity of the conjugatestowards the pathogenic cell population relative to the unconjugatedvinca compound.

The binding site for the ligand, such as a vitamin, can includereceptors for the ligand capable of specifically binding to the ligandwherein the receptor or other protein is uniquely expressed,overexpressed, or preferentially expressed by a population of pathogeniccells. A surface-presented protein uniquely expressed, overexpressed, orpreferentially expressed by the pathogenic cells is typically a receptorthat is either not present or present at lower concentrations onnon-pathogenic cells providing a means for selective elimination of thepathogenic cells. The drug delivery conjugates may be capable of highaffinity binding to receptors on cancer cells or other types ofpathogenic cells. The high affinity binding can be inherent to theligand or the binding affinity can be enhanced by the use of achemically modified ligand.

The drug delivery conjugates described herein can be administered in acombination therapy with any other known drug whether or not theadditional drug is targeted. Illustrative additional drugs include, butare not limited to, peptides, oligopeptides, retro-inversooligopeptides, proteins, protein analogs in which at least onenon-peptide linkage replaces a peptide linkage, apoproteins,glycoproteins, enzymes, coenzymes, enzyme inhibitors, amino acids andtheir derivatives, receptors and other membrane proteins, antigens andantibodies thereto, haptens and antibodies thereto, hormones, lipids,phospholipids, liposomes, toxins, antibiotics, analgesics,bronchodilators, beta-blockers, antimicrobial agents, antihypertensiveagents, cardiovascular agents including antiarrhythmics, cardiacglycosides, antianginals, vasodilators, central nervous system agentsincluding stimulants, psychotropics, antimanics, and depressants,antiviral agents, antihistamines, cancer drugs includingchemotherapeutic agents, tranquilizers, anti-depressants, H-2antagonists, anticonvulsants, antinauseants, prostaglandins andprostaglandin analogs, muscle relaxants, anti-inflammatory substances,stimulants, decongestants, antiemetics, diuretics, antispasmodics,antiasthmatics, anti-Parkinson agents, expectorants, cough suppressants,mucolytics, and mineral and nutritional additives.

In another illustrative aspect, the additional drug can be selected froma compound capable of stimulating an endogenous immune response.Suitable compounds include, but are not limited to, cytokines or immunecell growth factors such as interleukins 1-18, stem cell factor, basicFGF, EGF, G-CSF, GM-CSF, FLK-2 ligand, HILDA, MIP-1α, TGF-α, M-CSF,IFN-α, IFN-β, soluble CD23, LIF, and combinations thereof.

Therapeutically effective combinations of these immunostimulatoryfactors can be used. In one embodiment, for example, therapeuticallyeffective amounts of IL-2, for example, in amounts ranging from about0.1 MIU/m²/dose/day to about 15 MIU/m²/dose/day in a multiple dose dailyregimen, and IFN-α, for example, in amounts ranging from about 0.1MIU/m²/dose/day to about 7.5 MIU/m²/dose/day in a multiple dose dailyregimen, can be used along with the drug delivery conjugates toeliminate, reduce, or neutralize pathogenic cells in a host animalharboring the pathogenic cells (MIU=million international units;m²=approximate body surface area of an average human). In anotherembodiment IL-12 and IFN-α can be used in the above-describedtherapeutically effective amounts for interleukins and interferons, andin yet another embodiment IL-15 and IFN-α can be used in the abovedescribed therapeutically effective amounts for interleukins andinterferons. In an alternate embodiment IL-2, IFN-α or IFN-γ, and GM-CSFcan be used in combination in the above described therapeuticallyeffective amounts. Any other effective combination of cytokinesincluding combinations of other interleukins and interferons and colonystimulating factors can also be used.

Further, the additional drug can be any drug known in the art which iscytotoxic or cytostatic, enhances tumor permeability, inhibits tumorcell proliferation, promotes apoptosis, decreases anti-apoptoticactivity in target cells, is used to treat diseases caused by infectiousagents, enhances an endogenous immune response directed to thepathogenic cells, or is useful for treating a disease state caused byany type of pathogenic cell. Exemplary suitable additional drugs includeadrenocorticoids and corticosteroids, alkylating agents, antiandrogens,antiestrogens, androgens, aclamycin and aclamycin derivatives,estrogens, antimetabolites such as cytosine arabinoside, purine analogs,pyrimidine analogs, and methotrexate, busulfan, carboplatin,chlorambucil, cisplatin and other platinum compounds, tamoxiphen, taxol,paclitaxel, paclitaxel derivatives, Taxotere®, cyclophosphamide,daunomycin, rhizoxin, T2 toxin, plant alkaloids, prednisone,hydroxyurea, teniposide, mitomycins, discodennolides, non-vincamicrotubule inhibitors, epothilones, tubulysin, cyclopropylbenz[e]indolone, seco-cyclopropyl benz[e]indolone, O-Ac-seco-cyclopropylbenz[e]indolone, bleomycin and any other antibiotic, nitrogen mustards,nitrosureas, colchicine, colchicine derivatives, allocolchicine,thiocolchicine, trityl cysteine, Halicondrin B, dolastatins such asdolastatin 10, amanitins such as α-amanitin, camptothecin, irinotecan,and other camptothecin derivatives thereof, geldanamycin andgeldanamycin derivatives, estramustine, nocodazole, MAP4, colcemid,vindesine, vinblastine, vincristine, catharanthine, vindoline,leurosine, vinorelbine, imidocarb, sibutramine, toltrazuril,vinblastinoic acid, maytansines and analogs and derivatives thereof,gemcitabine, inflammatory and proinflammatory agents, peptide andpeptidomimetic signal transduction inhibitors, and any otherart-recognized drug or toxin. Other drugs that can be used incombination therapies include penicillins, cephalosporins, vancomycin,erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycosideantibiotics, gentamicin, amphotericin B, acyclovir, trifluridine,ganciclovir, zidovudine, amantadine, ribavirin, and any otherart-recognized antimicrobial compound. Analogs or derivatives of any ofthe above-described additional drugs can also be used in combinationtherapies.

In another illustrative embodiment, pharmaceutical compositions areprovided. The pharmaceutical compositions comprise an amount of a drugdelivery conjugate effective to eliminate a population of pathogeniccells in a host animal when administered in one or more doses. The drugdelivery conjugate is preferably administered to the host animalparenterally, e.g., intradermally, subcutaneously, intramuscularly,intraperitoneally, intravenously, or intrathecally. Alternatively, thedrug delivery conjugate can be administered to the host animal by othermedically useful processes, such as orally, and any effective dose andsuitable therapeutic dosage form, including prolonged release dosageforms, can be used. Exemplary excipients useful for oral dosage formsinclude, but are not limited to, corn starch, gelatin, lactose,magnesium stearate, sodium bicarbonate, cellulose derivatives, andsodium starch glycolate.

Examples of parenteral dosage forms include aqueous solutions of theactive agent, in an isotonic saline, 5% glucose or other well-knownpharmaceutically acceptable liquid carriers such as liquid alcohols,glycols, esters, and amides. The parenteral dosage form in accordancewith this invention can be in the form of a reconstitutable lyophilizatecomprising the dose of the drug delivery conjugate. In one aspect of thepresent embodiment, any of a number of prolonged release dosage formsknown in the art can be administered such as, for example, thebiodegradable carbohydrate matrices described in U.S. Pat. Nos.4,713,249; 5,266,333; and 5,417,982, the disclosures of which areincorporated herein by reference, or, alternatively, a slow pump (e.g.,an osmotic pump) can be used.

The additional drug in the combination therapy can be administered tothe host animal prior to, after, or at the same time as the drugdelivery conjugates and the additional drug can be administered as partof the same composition containing the drug delivery conjugate or aspart of a different composition than the drug delivery conjugate. Anysuch combination therapy at an effective dose of the additional drug canbe used.

In another illustrative aspect, more than one type of drug deliveryconjugate can be used. For example, the host animal can be treated in aco-dosing protocol with conjugates with different ligands such as, forexample, folate-vinca and vitamin B₁₂-vinca conjugates in combination,and the like. In another illustrative embodiment, the host animal can betreated with conjugates comprising more than one ligand such as, forexample, multiple folates or multiple vitamin B₁₂ molecules in oneconjugate, or combinations of ligands in the same conjugate such as avinca compound conjugated to both folate and vitamin B₁₂ ligands.Furthermore, drug delivery conjugates with different types of vincacompounds in separate drug delivery conjugates can be used.

The unitary daily dosage of the drug delivery conjugate can varysignificantly depending on the host condition, the disease state beingtreated, the molecular weight of the conjugate, its route ofadministration and tissue distribution, and the possibility of co-usageof other therapeutic treatments such as radiation therapy or additionaldrugs in combination therapies. The effective amount to be administeredto a host animal is based on body surface area, weight, and physicianassessment of patient condition. Effective doses can range, for example,from about 1 ng/kg to about 1 mg/kg, from about 1 μg/kg to about 500μg/kg, and from about 1 μg/kg to about 100 μg/kg.

Any effective regimen for administering the drug delivery conjugates canbe used. For example, the drug delivery conjugates can be administeredas single doses, or can be divided and administered as a multiple-dosedaily regimen. Further, a staggered regimen, for example, one to threedays per week can be used as an alternative to daily treatment, and forthe purpose of defining this invention such intermittent or staggereddaily regimen is considered to be equivalent to every day treatment andis comtemplated. In one illustrative embodiment the host animal istreated with multiple injections of the drug delivery conjugate toeliminate the population of pathogenic cells. In one embodiment, thehost is injected multiple times (preferably about 2 up to about 50times) with the drug delivery conjugate, for example, at 12-72 hourintervals or at 48-72 hour intervals. Additional injections of the drugdelivery conjugate can be administered to the host animal at an intervalof days or months after the initial injections(s) and the additionalinjections can prevent recurrence of the disease state caused by thepathogenic cells.

In one illustrative aspect, vitamins, or analogs or derivatives thereof,that can be used in the drug delivery conjugates include those that bindto receptors expressed specifically on activated macrophages, such asthe folate receptor which binds folate, or an analog or derivativethereof. The folate-linked conjugates, for example, can be used to killor suppress the activity of activated macrophages that cause diseasestates in the host. Such macrophage targeting conjugates, whenadministered to a host animal suffering from an activatedmacrophage-mediated disease state, work to concentrate and associate theconjugated vinca compounds in the population of activated macrophages tokill the activated macrophages or suppress macrophage function.Elimination, reduction, or deactivation of the activated macrophagepopulation works to stop or reduce the activated macrophage-mediatedpathogenesis characteristic of the disease state being treated.Exemplary of diseases known to be mediated by activated macrophagesinclude rheumatoid arthritis, ulcerative colitis, Crohn's disease,psoriasis, osteomyelitis, multiple sclerosis, atherosclerosis, pulmonaryfibrosis, sarcoidosis, systemic sclerosis, organ transplant rejection(GVHD) and chronic inflammations. Administration of the drug deliveryconjugate is typically continued until symptoms of the disease state arereduced or eliminated.

The drug delivery conjugates administered to kill activated macrophagesor suppress the function of activated macrophages can be administeredparenterally to the host animal, for example, intradermally,subcutaneously, intramuscularly, intraperitoneally, or intravenously incombination with a pharmaceutically acceptable carrier. Alternatively,the drug delivery conjugates can be administered to the host animal byother medically useful procedures and effective doses can beadministered in standard or prolonged release dosage forms. Thetherapeutic method can be used alone or in combination with othertherapeutic methods recognized for treatment of disease states mediatedby activated macrophages.

The following illustrative exemplified embodiments are not intended andshould not be construed as limiting. For example, in each compoundpresented herein, the stereochemistry of amino acids used in forming thelinker may b optionally selected from the natural L configuration, orthe unnatural D configuration. Each Example was characterized by NMR,MS, and/or UV spectroscopy, and/or HPLC as indicated; selectedcharacteristic signals are noted as appropriate.

METHOD EXAMPLES Method Example 1 Inhibition of Tumor Growth in Mice

The anti-tumor activity of the compounds described herein, whenadministered intravenously (i.v.) to tumor-bearing animals, wasevaluated in Balb/c mice bearing subcutaneous M109 tumors. Approximately11 days post tumor inoculation in the subcutis of the right axilla with1×10⁶ M109 cells (average tumor volume at t_(o)=60 mm³), mice (5/group)were injected i.v. three times a week (TIW), for 3 weeks with 1500nmol/kg of the drug delivery conjugate or with an equivalent dose volumeof PBS (control). Tumor growth was measured using calipers at 2-day or3-day intervals in each treatment group. Tumor volumes were calculatedusing the equation V=a×b²/2, where “a” is the length of the tumor and“b” is the width expressed in millimeters.

Method Example 2 Inhibition of Tumor Growth in Mice

The anti-tumor activity of the compounds described herein, whenadministered intravenously (i.v.) to tumor-bearing animals, wasevaluated in nu/nu mice bearing subcutaneous KB tumors. Approximately 8days post tumor inoculation in the subcutis of the right axilla with1×10⁶ KB cells (average tumor volume at t_(o)=50-100 mm³), mice(5/group) were injected i.v. three times a week (TIW), for 3 weeks with5 mmol/kg of the drug delivery conjugate or with an equivalent dosevolume of PBS (control). Tumor growth was measured using calipers at2-day or 3-day intervals in each treatment group. Tumor volumes werecalculated using the equation V=a×b²/2, where “a” is the length of thetumor and “b” is the width expressed in millimeters.

Method Example 3 Inhibition of Cellular DNA Synthesis

The compounds described herein were evaluated using an in vitrocytotoxicity assay that predicts the ability of the drug to inhibit thegrowth of folate receptor-positive KB cells. The compounds werecomprised of folate linked to a respective chemotherapeutic drug, asprepared according to the protocols described herein. The KB cells wereexposed for up to 7 h at 37° C. to the indicated concentrations offolate-drug conjugate in the absence or presence of at least a 100-foldexcess of folic acid. The cells were then rinsed once with fresh culturemedium and incubated in fresh culture medium for 72 hours at 37° C. Cellviability was assessed using a ³H-thymidine incorporation assay.

As shown in the figures herein, dose-dependent cytotoxicity wasmeasurable, and in most cases, the Ic₅₀ values (concentration of drugconjugate required to reduce ³H-thymidine incorporation into newlysynthesized DNA by 50%) were in the low nanomolar range. Furthermore,the cytotoxicities of these conjugates were reduced in the presence ofexcess free folic acid, indicating that the observed cell killing wasmediated by binding to the folate receptor.

Method Example 4 Relative Affinity Assay

The affinity for folate receptors (FRs) relative to folate wasdetermined according to a previously described method (Westerhof, G. R.,J. H. Schornagel, et al. (1995) Mol. Pharm. 48: 459-471) with slightmodification. Briefly, FR-positive KB cells were heavily seeded into24-well cell culture plates and allowed to adhere to the plastic for 18h. Spent incubation media was replaced in designated wells withfolate-free RPMI (FFRPMI) supplemented with 100 nM ³H-folic acid in theabsence and presence of increasing concentrations of test article orfolic acid. Cells were incubated for 60 min at 37° C. and then rinsed 3times with PBS, pH 7.4. Five hundred microliters of 1% SDS in PBS, pH7.4, were added per well. Cell lysates were then collected and added toindividual vials containing 5 mL of scintillation cocktail, and thencounted for radioactivity. Negative control tubes contained only the³H-folic acid in FFRPMI (no competitor). Positive control tubescontained a final concentration of 1 mM folic acid, and CPMs measured inthese samples (representing non-specific binding of label) weresubtracted from all samples. Notably, relative affinities were definedas the inverse molar ratio of compound required to displace 50% of³H-folic acid bound to the FR on KB cells, and the relative affinity offolic acid for the FR was set to 1.

Method Example 5 4T-1 Tumor Volume Assay

Six to seven week-old mice (female Balb/c strain) were obtained fromHarlan, Inc., Indianapolis, Ind. The mice were maintained on Harlan'sfolate-free chow for a total of three weeks prior to the onset of andduring this experiment. Folate receptor-negative 4T-1 tumor cells (1×10⁶cells per animal) were inoculated in the subcutis of the right axilla.Approximately 5 days post tumor inoculation when the 4T-1 tumor averagevolume was ˜100 mm³, mice (5/group) were injected i.v. three times aweek (TIW), for 3 weeks with 3 μmol/kg of drug delivery conjugate orwith an equivalent dose volume of PBS (control). Tumor growth wasmeasured using calipers at 2-day or 3-day intervals in each treatmentgroup. Tumor volumes were calculated using the equation V=a×b²/2, where“a” is the length of the tumor and “b” is the width expressed inmillimeters.

Method Example 6 Weight Determination

The percentage weight change of the mice was determined in mice (5mice/group) on the indicated days post-tumor inoculation (PTI) as shownin the graph for the samples described in the related tumor volumeassay.

Method Example 7 General Preparation of Folate-Peptides

Linkers described herein that include a peptide are prepared bypolymer-supported sequential approach using standard methods, such asthe Fmoc-strategy on an acid-sensitive Fmoc-AA-Wang resin.Illustratively, the folate-containing peptidyl fragmentPte-Glu-(AA)_(n)-NH(CHR₂)CO₂H (3) is prepared by the method shown inScheme 1 from Wang resin supported amino acids and Fmoc protected aminoacid synthesis.

In this illustrative embodiment of the processes described herein, R₁ isFmoc, R₂ is the desired appropriately-protected amino acid side chain,Wang is a 2-chlorotrityl-Resin, and DIPEA is diisopropylethylamine.Standard coupling procedures, such as PyBOP and others described hereinor known in the art are used, where the coupling agent is illustrativelyapplied as the activating reagent to ensure efficient coupling. Fmocprotecting groups are removed after each coupling step under standardconditions, such as upon treatment with piperidine, tetrabutylammoniumfluoride (TBAF), and the like. Appropriately protected amino acidbuilding blocks, such as Fmoc-Glu-OtBu, N¹⁰-TFA-Pte-OH, and the like,are used, as described in Scheme 1, and represented in step (b) byFmoc-AA-OH. Thus, AA refers to any amino acid starting material, that isappropriatedly protected. It is to be understood that the term aminoacid as used herein is intended to refer to any reagent having both anamine and a carboxylic acid functional group separated by one or morecarbons, and includes the naturally occurring alpha and beta aminoacids, as well as amino acid derivatives and analogs of these aminoacids. In particular, amino acids having side chains that are protected,such as protected serine, threonine, cysteine, aspartate, and the likemay also be used in the folate-peptide synthesis described herein.Further, gamma, delta, or longer homologous amino acids may also beincluded as starting materials in the folate-peptide synthesis describedherein. Further, amino acid analogs having homologous side chains, oralternate branching structures, such as norleucine, isovaline, β-methylthreonine, β-methyl cysteine, β,β-dimethyl cysteine, and the like, mayalso be included as starting materials in the folate-peptide synthesisdescribed herein.

The coupling sequence (steps (a) & (b)) involving Fmoc-protected aminoacids (AA) of the formula Fmoc-AA-OH is performed “n” times to preparesolid-support peptide (2), where n is an integer and may equal 0 toabout 100. Following the last coupling step, the remaining Fmoc group isremoved (step (a)), and the peptide is sequentially coupled to aglutamate derivative (step (c)), deprotected, and coupled toTFA-protected pteroic acid (step (d)). Subsequently, the peptide iscleaved from the polymeric support upon treatment with trifluoroaceticacid, ethanedithiol, and triisopropylsilane (step (e)). These reactionconditions result in the simultaneous removal of the t-Bu, t-Boc, andTrt protecting groups that may form part of the appropriately-protectedamino acid side chain. The TFA protecting group is removed upontreatment with base (step (f)) to provide the folate-containing peptidylfragment (3).

COMPOUND EXAMPLES Example 1

According to the general procedure of Method Example 7 (Scheme 1), Wangresin bound 4-methoxytrityl (MTT)-protected Cys-NH₂ was reactedaccording to the following sequence: 1) a. Fmoc-Asp(OtBu)-OH, PyBOP,DIPEA; b. 20% Piperidine/DMF; 2) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b.20% Piperidine/DMF; 3) a. Fmoc-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 4) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 5) a. Fmoc-Glu-OtBu, PyBOP, DIPEA; b. 20%Piperidine/DMF; 6) N¹⁰-TFA-pteroic acid, PyBOP, DIPEA. The MTT, tBu, andPbf protecting groups were removed with TFA/H₂O/TIPS/EDT(92.5:2.5:2.5:2.5), and the TFA protecting group was removed withaqueous NH₄OH at pH=9.3. Selected ¹H NMR. (D₂O) δ (ppm) 8.68 (s, 1H, FAH-7), 7.57 (d, 2H, J=8.4 Hz, FA H-12 &16), 6.67 (d, 2H, J=9 Hz, FA H-13& 15), 4.40-4.75 (m, 5H), 4.35 (m, 2H), 4.16 (m, 1H), 3.02 (m, 2H),2.55-2.95 (m, 8H), 2.42 (m, 2H), 2.00-2.30 (m, 2H), 1.55-1.90 (m, 2H),1.48 (m, 2H); MS (ESI, m+H⁺) 1046.

Example 2

According to the general procedure of Method Example 7 (Scheme 1), Wangresin bound 4-methoxytrityl (MTT)-protected Cys-NH₂ was reactedaccording to the following sequence: 1) a.Fmoc-β-aminoalanine(NH-MTT)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 2)a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 3) a.Fmoc-Asp(OtBu)—OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 4) a.Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 5) a.Fmoc-Glu-OtBu, PyBOP, DIPEA; b. 20% Piperidine/DMF; 6) N¹⁰-TFA-pteroicacid, PyBOP, DIPEA. The MTT, tBu, and TFA protecting groups were removedwith a. 2% hydrazine/DMF; b. TFA/H₂O/TIPS/EDT (92.5:2.5:2.5:2.5). Thereagents shown in the following table were used in the preparation:

Reagent (mmol) equivalents Amount H-Cys(4-methoxytrityl)-2- 0.56 1  1.0g chlorotrityl-Resin (loading 0.56 mmol/g) Fmoc-β-aminoalanine(NH- 1.122 0.653 g MTT)-OH Fmoc-Asp(OtBu)—OH 1.12 2 0.461 g Fmoc-Asp(OtBu)—OH1.12 2 0.461 g Fmoc-Asp(OtBu)—OH 1.12 2 0.461 g Fmoc-Glu-OtBu 1.12 20.477 g N¹⁰TFA-Pteroic Acid 0.70 1.25 0.286 g (dissolve in 10 ml DMSO)DIPEA 2.24 4  0.390 mL PyBOP 1.12 2 0.583 g

The coupling step was performed as follows: In a peptide synthesisvessel add the resin, add the amino acid solution, DIPEA, and PyBOP.Bubble argon for 1 hr. and wash 3× with DMF and IPA. Use 20% piperidinein DMF for Fmoc deprotection, 3× (10 min), before each amino acidcoupling. Continue to complete all 6 coupling steps. At the end wash theresin with 2% hydrazine in DMF 3× (5 min) to cleave TFA protecting groupon Pteroic acid.

Cleave the peptide analog from the resin using the following reagent,92.5% (50 ml) TFA, 2.5% (1.34 ml) H₂O, 2.5% (1.34 ml)Triisopropylsilane, 2.5% (1.34 ml) ethanedithiol, the cleavage step wasperformed as follows: Add 25 ml cleavage reagent and bubble for 1.5 hr,drain, and wash 3× with remaining reagent. Evaporate to about 5 mL andprecipitate in ethyl ether. Centrifuge and dry. Purification wasperformed as follows: Column-Waters NovaPak C₁₈ 300×19 mm; Buffer A=10mM Ammonium Acetate, pH 5; B=CAN; 1% B to 20% B in 40 minutes at 15ml/min, to 350 mg (64%); HPLC-RT 10.307 min., 100% pure, ¹H HMR spectrumconsistent with the assigned structure, and MS (ES—): 1624.8, 1463.2,1462.3, 977.1, 976.2, 975.1, 974.1, 486.8, 477.8.

Example 3

According to the general procedure of Method Example 7 (Scheme 1), Wangresin bound 4-methoxytrityl (MTT)-protected Cys-NH₂ was reactedaccording to the following sequence: 1) a.Fmoc-β-aminoalanine(NH-IvDde)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF;2) a. Fmoc-Asp(OtBu)—OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 3) a.Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 4) a.Fmoc-Asp(OtBu)—OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 5) a.Fmoc-Glu-OtBu, PyBOP, DIPEA; b. 20% Piperidine/DMF; 6) N¹⁰-TFA-pteroicacid, PyBOP, DIPEA. The MTT, tBu, and TFA protecting groups were removedwith a. 2% hydrazine/DMF; b. TFA/H₂O/TIPS/EDT (92.5:2.5:2.5:2.5). Thereagents shown in the following table were used in the preparation:

Reagent (mmol) Equivalents Amount H-Cys(4-methoxytrityl)-2- 0.56 1  1.0g chlorotrityl-Resin (loading 0.56 mmol/g) Fmoc-β-aminoalanine(NH- 1.122 0.596 g IvDde)-OH Fmoc-Asp(OtBu)—OH 1.12 2 0.461 g Fmoc-Asp(OtBu)—OH1.12 2 0.461 g Fmoc-Asp(OtBu)—OH 1.12 2 0.461 g Fmoc-Glu-OtBu 1.12 20.477 g N¹⁰TFA-Pteroic Acid 0.70 1.25 0.286 g (dissolve in 10 ml DMSO)Fm-Thiopropionic acid 0.70 1.25 199.08 DIPEA 2.24 4  0.390 mL PyBOP 1.122 0.583g

The coupling step was performed as follows: In a peptide synthesisvessel add the resin, add the amino acid solution in DMF, DIPEA, andPyBOP. Bubble argon for 1 hr. and wash 3×10 mL with DMF and IPA. Use 20%piperidine in DMF for Fmoc deprotection, 3×10 mL (10 min), before eachamino acid coupling. Continue to complete 6 coupling steps. At the endwash the resin with 2% hydrazine in DMF 3×10 mL (5 min) to cleave TFAprotecting group on Pteroic acid and IvDde protecting group onβ-aminoalanine. Finally, couple the free amine of the β-aminoalaninewith the Fmoc-thiopropionic acid in DMF using DIPEA and PyBop. Bubbleargon for 1 hr. and wash 3×10 mL with DMF and IPA. Dry the resin underargon for 30 min.

Cleave the peptide analog from the resin using the following reagent,92.5% (50 ml) TFA, 2.5% (1.34 ml) H₂O, 2.5% (1.34 ml)Triisopropylsilane, 2.5% (1.34 ml) ethanedithiol, the cleavage step wasperformed as follows: Add 25 ml cleavage reagent and bubble for 1.5 hr,drain, and wash 3× with remaining reagent. Evaporate to about 5 mL andprecipitate in ethyl ether. Centrifuge and dry. Purification wasperformed as follows: Column-Waters NovaPak C₁₈ 300×19 mm; Buffer A=10mM Ammonium Acetate, pH 5; B=CAN; 1% B to 20% B in 40 minutes at 15ml/min, to 450 mg (65%); ¹H HMR spectrum consistent with the assignedstructure.

Example 4

According to the general procedure of Method Example 7 (Scheme 1), Wangresin bound MTT-protected Cys-NH₂ was reacted according to the followingsequence: 1) a. Fmoc-Asp(OtBu)—OH, PyBOP, DIPEA; b. 20% Piperidine/DMF;2) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 3) a.Fmoc-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 4) a.Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 5) a.Fmoc-Glu(γ-OtBu)—OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 6)N¹⁰-TFA-pteroic acid, PyBOP, DIPEA. The MTT, tBu, and Pbf protectinggroups were removed with TFA/H₂O/TIPS/EDT (92.5:2.5:2.5:2.5), and theTFA protecting group was removed with aqueous NH₄OH at pH=9.3. The ¹HNMR spectrum was consistent with the assigned structure.

Example 5

According to the general procedure of Method Example 7 (Scheme 1), Wangresin bound MTT-protected D-Cys-NH₂ was reacted according to thefollowing sequence: 1) a. Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 2) a. Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 3) a. Fmoc-D-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 4) a. Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 5) a. Fmoc-D-Glu-OtBu, PyBOP, DIPEA; b. 20%Piperidine/DMF; 6) N¹⁰-TFA-pteroic acid, PyBOP, DIPEA. The MTT, tBu, andPbf protecting groups were removed with TFA/H₂O/TIPS/EDT(92.5:2.5:2.5:2.5), and the TFA protecting group was removed withaqueous NH₄OH at pH=9.3. The ¹H NMR spectrum was consistent with theassigned structure.

Example 6

2-[(Benzotriazole-1-yl-(oxycarbonyloxy)-ethyldisulfanyl]-pyridine HCl(601 mg) and 378 μL of DIPEA were sequentially added to a solution ofdesacetyl vinblastine hydrazide (668 mg) in 5 ml of DCM at 0° C. Thereaction was allowed to warm to room temperature and stirred for 3hours. TLC (15% MeOH in DCM) showed complete conversion. The mixture waspurified by silica gel chromatography (1:9 MeOH/DCM). The combinedfractions were evaporated, redissolved in DCM and washed with 10%Na₂CO₃, brine, dried (MgSO₄), and evaporated to 550 mg (80%); HPLC-RT12.651 min., 91% pure, ¹H HMR spectrum consistent with the assignedstructure, and MS (ESI+): 984.3, 983.3, 982.4, 492.4, 491.9, 141.8.Additional details of this procedure are described in U.S. patentapplication publication no. US 2005/0002942 A1, incorporated herein inits entirety by reference.

Example 7

Mitomycin C-ethyl disulfide propionic acid was prepared according to thefollowing scheme

To a solution of the aminoethyldisulfide propionic acid (81 mg, 0.372mmol) in 2 mL of methanol (MeOH) was added the DIPEA (0.13 mL, 0.746mmol). To this solution was slowly added the mitomycin-A (100 mg, 0.286mmol) in MeOH (3.0 mL). The resulting solution was allowed to stir for 3h. TLC analysis (20% MeOH in CHCl₃) indicated that the reaction wascomplete. The solvent was removed under reduced pressure and the residuewas purified using a silica column. Gradient elution (10% to 20% MeOH inCHCl₃/0.5% TEA gave pure fractions of the product (110 mg, 77%).Selected ¹H NMR signals (CDCl₃) δ (ppm) 3.50 (d, 1H), 3.56 (dd, 1H),3.90 (t, 2H), 4.15 (d, 1H), 4.25 (t, 1H), 4.68 (dd, 1H).

Example 8

Prepared according to the process of Example 7.

Example 9

In a polypropylene centrifuge bottle, Example 2 (82 mg, 0.084 mmol) wasdissolved in 5 mL of water and bubbled with argon for 10 min. In anotherflask, a 0.1N NaHCO₃ solution was argon bubbled for 10 min. pH of thelinker solution was adjusted to about 6.9 using the 0.1N NaHCO₃solution. The vinblastine hydrazide derivative (Example 6, 91 mg, 0.092mM) in 5 mL of tetrahydrofuran (THF) was added slowly to the abovesolution. The resulting clear solution was stirred under argon for 15min to 1 h. Progress of the reaction was monitored by analytical HPLC(10 mM ammonium acetate, pH=7.0 and acetonitrile). THF was evaporated,and the aqueous solution was filtered and injected on a prep-HPLC column(XTerra Column, 19×300 mM). Elution with 1 mM sodium phosphate pH=7.0and acetonitrile resulted in pure fractions containing the product,which was isolated after freeze-drying for 48 h (78 mg, 50%);C₈₃H₁₀₃N₁₉O₂₆S₂; exact mass: 1845.68; MW: 1846.95; HPLC-RT 15.113 min.,100% pure, ¹H HMR spectrum consistent with the assigned structure, andMS (ES−): 1846.6, 1845.5, 933.3, 924.2, 923.3, 922.5, 615.6, 614.7,525.0.

FIGS. 21A and 21B show the relative binding affinity for folate versusExample 9, and the effects of Example 9 on ³H-thymidine incorporation,the IC₅₀ of the conjugate (58 nM), and that folate competes with theconjugate for binding to the folate receptor demonstrating thespecificity of binding of the conjugate. The assays were conductedaccording to Method Examples 4 and 3, respectively.

FIG. 1B shows the activity of Example 9 on ³H-thymidine incorporation inKB cells with (∘) and without () excess folic acid; IC₅₀ of Example 9is about 58 nM.

Example 10

In a polypropylene centrifuge bottle, Example 3 (56 mg) was dissolved in7.5 mL of water and bubbled with argon for 10 min. In another flask, a0.1 N NaHCO₃ solution was bubbled with argon for 10 mM. The pH of theExample 3 solution was adjusted to 6.9 using the 0.1 N NaHCO₃ solution.Example 6 (44 mg) in 7.5 mL of tetrahydrofuran (THF) was added slowly tothe Example 3 solution. The resulting clear solution was stirred underargon for 15 min to 1 h. Progress of the reaction was monitored byanalytical HPLC (10 mM ammonium acetate, pH=7.0 and acetonitrile). THFwas evaporated and the aqueous solution was filtered and purified byprep-HPLC. Elution with 1 mM sodium phosphate pH=7.0 and acetonitrileresulted in pure fractions, which were pooled, evaporated at ambienttemperature, and the resulting aqueous solution was adjusted to pH 4.0using 0.1 N HCl. Example 10 was isolated after freeze-drying for 48 h(61 mg, 64%). ¹H HMR spectrum and LCMS data consistent with the assignedstructure.

Example 11

Method A. Example 11 was prepared according to the following process:

Mitomycin C-ethyl disulfide propionic acid (34.4 mg, 0.069 mmol) wasdissolved in dry THF (1 mL) under argon. N-hydroxy succinamide (7.9 mg,0.069 mmol) followed by dicyclohexyl carbodiimide (14.2 mg, 0.069 mmol)was added. Di-isopropylethylamine (0.024 mL, 0.138 mmol) was added andthe resulting mixture was stirred for 3 h. In a polypropylene centrifugebottle, vinblastine folate (Example 9, 26 mg, 0.014 mmol) was dissolvedin 3 mL of water. The pH of the solution was slowly adjusted to 8.5using 0.1 N NaHCO₃. The activated mitomycin C derivative prepared asdescribed herein was added to the folate solution as a 3 mL THFsolution. The resulting solution was stirred under argon for 15 min to 1h, where the progress of the reaction was monitored by analytical HPLC(10 mM ammonium acetate and acetonitrile, pH=7.0). The THF was removedunder reduced pressure and the aqueous solution was filtered andinjected onto a prep-HPLC column (X-terra Column, 19×300 mm). Elutionwith 1 mM sodium phosphate (pH=7.0) and acetonitrile resulted in purefractions, which were evaporated and freeze-dried for 48 h to 12 mg(50%, based on recovered starting material). ¹H NMR and mass spectraldata supported that assigned structure as shown in FIGS. 9 and 10respectively. C₁₀₃H₁₂₇N₂₃O₃₂S₄; Exact Mass 2325.79; MW 2327.51. HPLC-RT20.054 min., 99% pure, ¹H HMR spectrum consistent with the assignedstructure, and MS (ES+): 1552.5, 116.0, 1165.3, 1164.3, 1148.4, 744.9,746.4, 745.6.

Method B. Anhydrous DMF (4.5 mL) was syringed into a mixture of Example10 (103 mg, 48.7 μmol) and Example 8 (NO₂—PySSCH₂CH₂-MMC, 33.4 mg, 1.25eq) at room temperature under argon. To the resulting solution weresyringed in DIPEA (84.9 μL, 10 eq) and DBU (72.9 μL, 10 eq) in tandem.The reaction mixture was stirred at room temperature under argon for 20minutes, then transferred into a stirring diethyl ether (50 mL). Theresulting suspension was centrifuged, the precipitate was washed withdiethyl ether (15 mL×2), then dissolved in phosphate buffer (9 mL, 1.25mM, pH 6.8) and was subject to a preparative HPLC (Column: Waters XTerraRP 18, 7 μm, 19×300 mm; Mobile phases: A=1.25 in M phosphate buffer, pH6.8, B=acetonitrile; Method: 10% B to 40% B over 25 min at 25 mL/min).Fractions from 11.72-13.88 minutes were collected and freeze-dried toafford 105.8 mg material, containing 99.2 mg and 6.6 mg phosphate salts.

Method C. Example 11 was prepared according to the following process in34% yield:

FIG. 2 shows the relative binding affinity for folic acid (, 1.0)versus Example 11 (▪, 0.21). The data in FIG. 2 shows that the conjugatehas high relative binding to the folate receptor. The assay wasconducted according to Method Example 4.

FIGS. 1B and 3 show the effects of Examples 9 (having a single drug) and11 (having a pair of drugs), respectively, on ³H-thymidineincorporation, the IC₅₀ of the conjugates of Example 9 (58 nM) and ofExample 11 (5 nM). The data in FIGS. 1B and 3 also show that folic acidcompetes with the conjugates for binding to the folate receptordemonstrating the specificity of binding of the conjugate. The assayswere conducted according to Method Example 3. In addition, Example 11having two drugs showed more than 10-fold more potency at the folatereceptor than Example 9 having only a single drug.

FIG. 4 shows the in vitro cytotoxic activity of Example 11 (a) on threedifferent tumor cell lines (KB, 4T-1cl2, and ID8-cl15). In addition,FIG. 4 shows that the cytotoxic activity of Example 11 reduced in thepresence of excess folic acid (b), indicating that Example 11 is actingat the folate receptor.

FIGS. 5A and 5B show the activity of Example 11 at two different doses(1 μmol/kg & 2 μmol/kg) against M109 lung cancer tumors in Balb/c miceand on the weight of Balb/c mice (Balb/c mice were used for the M109tumor volume assay). The assays were performed according to MethodExamples 1 and 6, respectively. Example 11 inhibited the growth of solidtumors, but had little effect on the weight of the mice at both doses.In addition, the higher dose (2 μmol/kg) showed strong inhibition oftumor growth, even after the dosing was terminated on day 20. Thevertical line corresponds to the last dosing day (Day 20). Five animalswere tested, and at the higher dose of 2 μmol/kg, all five animalsshowed a complete response.

FIG. 6 shows the activity of Example 11 at 1 μmol/kg TIW for 2 weeks onFR-positive KB tumors with (b) and without (c) 40 μmol/kg EC20 (rheniumcomplex), compared to controls (a). The vertical dashed line indicatesthe last dosing day. The figures show that Example 11 inhibits thegrowth of solid tumors, and that inhibitory effect is prevented(competed) by the EC20 rhenium complex. In addition, the figures showthat treatment with Example 11 did not affect the weight of the testanimal significantly from controls. EC20 (rhenium complex) is thecompound of the formula

chelated to Rhenium. The preparation of EC20 is described in U.S. patentapplication publication no. US 2004/0033195 A1, the synthetic proceduredescription of which is incorporated herein by reference. The assay wasperformed according the Method Example 2. EC20 acts as a competitor ofExample 11 at folate receptors, and the results show the specificity ofthe effects of Example 11.

FIG. 8 shows the activity of Example 11 at 1 μmol/kg TIW on folatereceptor positive s.c. implanted human xenograft KB tumors with (b) andwithout (c) added 40 μmol/kg EC20 (rhenium complex) in nude mice. Thedata in FIG. 8 show that Example 11 inhibits the growth of solid tumors,and that the inhibitory effect is prevented (competed against) by theEC20 rhenium complex, (b) versus (c). In addition, the data in FIG. 8show that treatment with Example 11 did not significantly affect theweight of the tested nude mice animal model compared to controls (a).

FIG. 10 shows the activity of Example 11 at 2 μmol/kg TIW (e) on folatereceptor positive human tumors in nude mice compared to a mixture of theunconjugated base drugs, mitomycin C and desacetylvinblastinemonohydrazide, at 0.5 μmol/kg TIW (b), 1 pmol/kg TIW (c), and 2 μmol/kgTIW (d), compared to untreated controls (a). The data in FIG. 10 showthat Example 11 inhibits the growth of solid tumors and gives a completeresponse in five out of five test animals. In contrast, treatment withthe mixture of base drugs at 0.5 μmol/kg TIW (b), or at 1 μmol/kg TIW(c) did not show a complete response in any of the five test animals.The high dose of the mixture of base drugs at 2 μmol/kg TIW (d) wasdiscontinued before day 20 due to observed toxicity, as shown in FIG. 11showing the effect of the base drugs and Example 11 on test animalweight.

FIG. 11 shows that Example 11 (e) did not significantly affect theweight of the test animals during treatment from controls (a). Incontrast to Example 11, the data in FIG. 11 show that prolongedtreatment with the lower doses of the mixture of the unconjugated basedrugs, mitomycin C and desacetylvinblastine monohydrazide, at (0.5μmol/kg TIW (b) and 1 μmol/kg TIW (c)) caused weight loss in testanimals that was significant compared to controls (a). In addition, thehigh dose (2 pmol/kg TIW (d)) of the mixture of the unconjugated basedrugs caused the greatest weight loss, leading to the termination ofthat test.

The compounds described herein may be useful in treating large orestablished tumors. Illustratively, Example 11 is effective on largetumors. FIG. 12 shows the activity of Example 11 at 2 μmol/kg TIW, 2weeks on large (250 mm³, 500 mm³, and 750 mm³) s.c. KB tumors. Treatmentwith Example 11 was initiated when the tumors reached one of the threetarget volumes, as indicated by the vertical arrows corresponding to thetumor volume. The data in FIG. 12 show that Example 11 inhibits thegrowth of large tumors and gives a complete response in test animals.

FIG. 13 shows the activity of Example 11 (e) at 1 μmol/kg TIW for twoweeks of treatment on established s.c. KB tumors, compared to controls(a); the conjugates of each single drug alone, mitomycin C conjugate (b)and desacetylvinblastine monohydrazide conjugate (c), or a mixture ofthose single drug conjugates (d). Each drug conjugate was dosed at thesame level of 1 μmol/kg TIW for two weeks of treatment. The figure showsthat Example 11 performs better than either single drug conjugate or amixture of both single drug conjugates. Surprisingly, the mixture ofsingle drug conjugates did not perform significantly better than thesingle drug conjugates dosed individually, and none of the single drugconjugate dosing regimens was statistically significant from thecontrols. Only the compound of Example 11 was superior to controls. Inaddition, these data suggest a synergistic effect of having both a vincadrug and a mitomycin drug on the single conjugate.

Examples 12 to 14

Prepared according to the processes and conditions described herein,including the processes described hereinabove for Example 11. Additionaldetails for the preparation of the required thiosulfonate orpyridyldithio-activated vinblastine, and maleimide-activated vinblastinederivatives are described in U.S. patent application publication no. US2005/0002942 A1. Additional details for the preparation of the requiredmitomycin derivatives are described in U.S. patent applicationpublication no. US 2005/0165227 A1, the disclosure of which isincorporated herein by reference.

Example 12

FIG. 15 shows the activity of Example 12 at 100 nM on ³H-thymidineincorporation into FR-positive KB cells versus the pulse time. The assaywas performed according to Method Example 3.

Example 13

Example 14

1. A receptor binding drug delivery conjugate comprising: (a) a receptorbinding moiety; (b) a polyvalent linker; and (c) two or more drugs, oranalogs or derivatives thereof; wherein the receptor binding moiety iscovalently linked to the polyvalent linker; the two or more drugs, oranalogs or derivatives thereof, are covalently linked to the polyvalentlinker; and the polyvalent linker comprises one or more releasablelinkers. 2.-52. (canceled)